Cyclin

Members of the cyclin family of proteins are key regulators of the cell cycle. Cyclins bind to and activate members of the
cyclin-dependent kinase (Cdk) family to effect cell cycle progression. Cell cycle progression is controlled by the relative
levels of individual cyclin family members. Progression through the G1->S->G2->M cycle follows successive oscillations
in the levels of cyclins, D, E, A and B. Cyclins and cyclin-dependent kinases were discovered by Leland H. Hartwell, R.
Timothy Hunt, and Paul M. Nurse, who thus won the 2001 Nobel Prize in Physiology or Medicine.
● Cyclin D family members are G1 phase cyclins that regulate the entry of cells into G1 from Go.
● Cyclin D is upregulated by growth factor and external signals through the Ras-GTPase signalling pathway.
● Cyclin D couples with Cdk4 and Cdk6.
● The cyclin-D-dependent kinases commit the cell to enter S-phase.
● Cyclin D-Cdk4 hypo-phosphorylates retinoblastoma protein (pRB) and facilitates the expression of cyclin E.
● Cyclin E and Cyclin A are able to bind Cdk2 and promote the cell cycle progression through G1/S transition.
● Cyclin E-Cdk2 and Cyclin A-Cdk2 hyper-phosphorylate and inactivate pRb. The inactivation of pRb leads to
activation of E2F transcription factors.
● Cyclin E stimulates replication complex assembly through interaction with Cdc6.
● Cyclin A activates DNA synthesis by the replication complex already assembled and inhibits assembly of new
replication complex.
● Cyclin E reinitiates the replication complex that is blocked by cyclin A.
● Cyclins B1 and B2 are M-phase cyclins. Cyclin B1, Cyclin B2 and their catalytic partner Cdk1 , are components
of the M phase/maturation promoting (MPF) factor that regulates processes that lead to assembly of the mitotic
spindle and sister-chromatid pair alignment on the spindle.
● G1 cyclins are ubiquitinated by the SCF complex, while mitotic cyclins are ubiquitinated by the anaphase-
promoting complex (APC).

Zinc Fingers
● A zinc finger is a specific protein domain that coordinates one or more zinc ions through its cysteine and/or
histidine residues to stabilize its structure.
● Zinc fingers are composed of both alpha helices and beta sheets. Their specificity for binding is encoded mainly
in the amino acid sequence of the alpha sheets.
● Zinc fingers are structurally diverse and exhibit a wide range of
functions, from DNA-RNA-binding to protein-protein interactions
and membrane association.
● There are more than 40 types of zinc fingers known today.
● Zinc fingers were originally discovered in the transcription factor
TFIIIA from frog eggs, which contains nine zinc fingers in a row.
● Zinc fingers are very highly conserved amongst many species.
● Zinc finger proteins of the C2H2-type are amongst the most
frequently occurring proteins in the genome of Homo sapiens.
Most C2H2-type zinc finger proteins are transcriptional activators
or repressors that bind DNA.
● Zinc finger nucleases (ZFNs) are an extremely powerful tool
based on the zinc finger motif design, which have recently been
synthesised to perform targeted genomic manipulation in a variety
of cell types. An on-going clinical trial is currently evaluating ZFNs that disrupt the CCR5 gene in CD4+ human
T-cells as a potential treatment for HIV/AIDS.
TATA box and TBP
One of the most important promoters for RNA polymerase II is a short consensus sequence known as the TATA box. It
helps define the start site for transcription and recruits the polymerase. The TATA box is located on the 5’ side of the start
site and is termed a ‘cis-acting’ element. The specific sequence is TATAAT. A mutation of a single base pair in this
sequence noticeably impairs its promoter activity.
The TATA box is often paired with an ‘initiator element’. This is a sequence found at the transcriptional start site between
positions -3 and +5. This sequence defines the start site and its presence increases transcriptional activity. Additional
regulatory sequences can be found between -40 and -150. These may include the CAAT box and the GC box.
Cis acting elements like the TATA box require Transcription factors to bind to them for the initiation of expression. In the
TATA box promoters, the key initial event is the recognition of the TATA box by the TATA-box-binding protein, (TBP).
The TBP is a 30kD protein which binds to the TATA sequence 10^5 times more strongly as to non-consensus sequences.
It is a saddle shaped protein consisting of two similar domains. The TATA box sequence binds to the concave surface of
the TBP which induces large conformational changes in the bound DNA. The double helix is substantially unwound to
widen its minor groove enabling it to make extensive contact with the concave side of the TBP. Hydrophobic interactions
are prominent at this interface.
The TBP-TATA complex is distinctly asymmetric which is crucial for specifying a unique start site and ensuring that
transcription proceeds unidirectionally. The TBP-TATA complex is the heart of the initiation complex in eukaryotes.
Other TFs join onto this complex to form the basal transcription apparatus.

Glucose
 Glucose is a monosaccharide that is central to all life.
 Glucose is the main product of photosynthesis.
 Glucose exists in several different molecular structures, but all of these structures can be divided into two
stereoisomers, L and D. Only one set of these isomers exists in nature, those derived from the "right-handed form" of
glucose, D-glucose. D-glucose is often referred to as ‘dextrose’.
 Glucose is used as an energy source in almost most organisms in the form of A) Aerobic respiration B) Anaerobic
respiration or C) Fermentation, all of which begin with glycolysis.
 The first step of glycolysis is the phosphorylation of glucose by the enzyme hexokinase to prepare it for further
breakdown, providing a net of 2 molecules of ATP per molecule of Glucose.
 Glucose provides approximately 3.75 kilocalories (16 kilojoules) of energy in the form of ATP per gram.
 Insulin is a hormone that regulates the concentration of glucose in the blood. A high fasting blood sugar level is an
indication of pre-diabetic/diabetic conditions.
 Glucose is critical in the production of proteins and in lipid metabolism.
 Glucose is a precursor for vitamin C (ascorbic acid).
 Glucose and cancer: Cancer cells exhibit a high rate of glucose consumption, beyond that necessary for ATP
synthesis. This is because glucose aids in the generation of biomass and regulates cellular signaling, both of which are
critical for oncogenic progression. A key rate-limiting step in glucose utilization is the transport of glucose across the
plasma membrane by glucose transporters (GLUTs).The identification and targeting of tumor-specific GLUTs could
provide a promising approach to block glucose-regulated metabolism and signalling more comprehensively in
cancers.
 The Warburg effect is the observation that most cancer cells predominantly produce energy by a high rate of
glycolysis followed by lactic acid fermentation in the cytosol, rather than by a comparatively low rate of glycolysis
followed by oxidation of pyruvate in mitochondria as in most normal cells. The Warburg effect may simply be a
consequence of damage to the mitochondria in cancer, or an adaptation to low-oxygen environments within tumours,
or a result of cancer genes shutting down the mitochondria because they are involved in the cell's apoptosis program
which would otherwise kill cancerous cells.
 One means of testing for the presence of glucose is using Benedict’s solution.
 cAMP
● cAMP (Cyclic Adenosine MonoPhosphate) is an important second messenger produced from ATP by the enzyme Adenylate
Cyclase.
● It is an intracellular molecule that changes in concentration in response to environmental signals such as a hormone (e.g.
adrenalin) binding to it’s receptor (B2-AR).
● The function of cAMP is to relay information from receptor-ligand complexes.
● A major advantage of cAMP as a secondary messenger is its potential to amplify a signal. In the B-AR pathway for instance,
an increased concentration of cAMP can result from the binding of a single molecule of adrenalin to its receptor. The many
cAMP molecules produced can then go on to affect a wide array of cellular activities such as the production of ATP in
muscle, enhancement of storage fuel (such as glycogen) degradation and opening of chloride channels.
● Most of these effects of cAMP in eukaryotes are mediated by its activation of Protein Kinase A.
● PKA consists of two regulatory chains (r2) and two catalytic chains (c2). In the absence of cAMP, the r2c2 complex in PKA
is catalytically inactive. The binding of cAMP to the regulatory chains releases the catalytic chains rendering them active.
● Activated PKA phosphorylates specific serine and threonine residues in their target cells.
● The result of cAMP signalling may be effects as diverse as Ca2+ influx, excitability, gene expression, as well as cell-specific
processes such as glycogenolysis and lipolysis.
● Phosphodiesterase hydrolyses cAMP into AMP.

Cytochrome C
Cytochrome c is a water-soluble protein found in the inner mitochondrial membrane. It is approximately 13 kDa in size. It
plays important roles in oxidative phosphorylation and apoptosis.
As an electron carrier in oxidative phosphorylation, cytochrome c shuttles four electrons, one at time, via its heme group
from Complex III (cytochrome c reductase) to Complex IV (cytochrome c oxidase).
As a crucial player in apoptosis, cytochrome c is released from the mitochondria to the cytosol where it binds to an
adaptor subunit, APAF-1 in the presence of dATP. This leads to activation of caspase 9. Caspase 9 triggers activation of
other caspases which cleave and destroy other proteins, resulting in cell death via apoptosis.
The release of cytochrome c into the cytosol and cytochrome-c-mediated apoptosis are controlled by multiple layers of
regulation, the most prominent players being members of the B-cell lymphoma protein-2 (BCL2) family.
The mobilization of cytochrome can be amplified by the further production of ROS.

PCR
PCR (Polymerase Chain Reaction) is a revolutionary method developed by Kary Mullis in the 1980s. It is based on using
the ability of DNA polymerase to synthesize new strand of DNA complementary to the offered ‘template strand’. Because
DNA polymerase can add a nucleotide only onto a pre-existing 3'-OH group, it needs a primer to which it can add the first
nucleotide. This requirement makes it possible to target a specific region of template sequence that the researcher wants to
amplify. At the end of the PCR reaction, the specific sequence will be accumulated in billions of copies.
PCR reactions require the following components:
DNA template - the sample DNA that contains the target sequence. At the beginning of the reaction, high temperature is
applied to the original double-stranded DNA molecule to separate the strands from each other.
DNA polymerase - a type of enzyme that synthesizes new strands of DNA complementary to the target sequence. The
first and most commonly used of these enzymes is Taq DNA polymerase (from Thermis aquaticus). Taq polymerase has
the desirable quality of being heat resistant.
Primers - short pieces of single-stranded DNA that are complementary to the target sequence. The polymerase begins
synthesizing new DNA from the end of the primer.
Nucleotides (dNTPs - deoxynucleotide triphosphates) - single units of the bases A, T, G, and C, which are essentially
"building blocks" for new DNA strands.
RT-PCR (Reverse Transcription PCR) is PCR preceded with conversion of sample RNA into cDNA with the enzyme
reverse transcriptase
Applications of PCR: Cloning, genetic engineering, genetic sequencing.

P53
P53 is a tumor suppressor gene. Its main functions include: A) Activation of DNA repair enzymes in response to DNA
damage B) Induction of cell cycle growth arrest and C) Initiation of apoptosis if DNA damage proves to be irreparable.
Because of its so called ‘guardian of the genome’ function, p53 the most commonly mutated gene in all cancers (50%).
The p53 protein is encoded by the Tp53 gene. Other biological functions of the p53 protein include regulation of
senescence, DNA metabolism, angiogenesis, cellular differentiation, and the immune response.
During p53 activation, the half-life of the protein is increased allowing for its accumulation in stressed cells. It gets
phosphorylated on its N-terminal domain, inducing a conformational change. (The N-terminal transcriptional activation
domain contains a large number of phosphorylation sites and can be considered as the primary target for protein kinases
transducing stress signals.)
Depending on the type of cellular stress, p53 can induce: A) G1 arrest through activation of the cyclin-dependent kinase
inhibitor p21 B) Regulation of the G2/M transition by inhibition of Cdc2. (Cdc2 needs to bind to cyclin B1 in order to
function. Repression of cyclin B1 by p53 arrests of cells in G2.)
MDM2 is an E3 ubiquitin ligase which controls p53 degradation. Many tumors overexpress MDM2, even tumors without
p53 mutations. Targeting MDM2 for p53 stabilization seems to be promising for cancer therapy.
The first p53-based gene therapy was reported in 1996. A retroviral vector containing the wild-type p53 gene under the
control of an actin promoter was injected directly into tumors of non-small cell lung cancer patients.
Since most human cancers harbour mutated p53, the restoration of p53 for cancer therapy is very attractive. PRIMA-1 is a
small molecule recently identified by cell-based screening which restores sequence-specific DNA binding and the active
conformation of p53.

MHC complexes
Class I MHC proteins are expressed on all nucleated cell surfaces. They consist of only one chain. They are highly
conserved and highly polymorphic. Their function is to help present antigens to cytotoxic T lymphocytes (CTLs, CD8+ T
cells).
Most CTLs possess both T-cell receptors (TCR) and CD8+ molecules on their surfaces. The TCRs recognize peptides
when they are expressed in complexes with MHC Class I molecules. For the TCR to bind a peptide-MHC complex the
TCR must have a structure which allows it to bind the peptide-MHC complex. The accessory molecule CD8, must bind to
the alpha-3 domain of the MHC Class I molecule. Due to genetic recombination events, each CTL expresses a unique
TCR which only binds a specific MHC-peptide complex. CTLs which recognize self-peptides (i.e. peptides produced by
the normal host body as opposed to foreign or cancerous cells) are removed in the thymus or tolerized after their release
from the thymus.

The MHC Class II proteins are expressed only on B lymphocytes, macrophages, and other APCs. They consist of 2
chains; alpha and beta, allowing for even more variability. Their primary function is to present peptides which have been
 digested from external sources to CD4+ cells. They are also needed for T-cell communication with B-cells and
macrophages. Class II MHC molecules present their bound antigens to T-helper cells.
The major histocompatibility complex is a gene dense region located on chromosome 6 in humans. It contains more than
200 genes known as Human Leukocyte Antigen genes or HLA genes. There are three sub-classes of HLA I genes in
humans: HLA -A, -B, and -C. There are also three pairs of MHC class II α- and β-chain genes, called HLA-DR, -DP, and
-DQ. Because of the polygeny of the MHC, every person will express at least three different antigen-presenting MHC
class I molecules and three (or sometimes four) MHC class II molecules on his/her cells.
The HLA genes harbour many genes associated with the immune system and histones. Classically it it 4mb in size.

Cytochrome p450
● Cytochromes P450 (CYPs) are haeme-containing membrane proteins in the endoplasmic reticulum (ER) or mitochondrial
inner membrane.
● They require a source of electrons from an electron transfer chain to function: (NADPH-cytochrome P450 oxidoreductase
(POR) in the ER and ferredoxin (FDX) plus ferredoxin reductase (FDXR) in mitochondria).
● CYP functions can be divided into oxidation/reduction of endogenous or exogenous compounds (e.g. drugs, chemicals,
pollutants, etc.).
● Endogenous P450 substrates are mainly lipids including steroids, lanosterol, bile acids, vitamin D, retinoic acid, eicosanoids,
fatty acids and some haeme breakdown products.
● Cytochromes P450 in humans (and most vertebrates) are divided into 18 CYP gene families based on sequence similarity.
● Polymorphism in CYP genes can alter a person's ability to metabolise drugs (poor, intermediate, efficient or ultra-rapid
metaboliser).
● CYP2D6 and CYP3A4 are the major drug-metabolising enzymes in humans. CYP2D6 appears to be the most polymorphic.
● Alterations in CYP drug metabolism are often responsible for adverse drug reactions – especially when multiple drugs are
taken.
● The primary carcinogenic component of tobacco is the polycystic aromatic hydrocarbons. These are stable organic
molecules that cause DNA damage. Cytochrome p450 initiates their break down through the introduction of reactive polar
groups. This conjugation allows other enzymes such as Glutathione S transferase to further modify them so that efflux
pumps are able to eliminate them from our bodies. GSTM1 (Gluthathione-S-Transferase) is lacking in 50% of caucasians.
Null individuals have a reduced ability to detoxify these toxins and hence have a higher risk to developing lung cancer.

Phosphatidylcholine (PtdCho)
Phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine are important phospholipids which get
hydrolysed by the enzyme phospholipase A2, allowing for the liberation of arachidonic acid.
PtdCho is the principal phospholipid in mammalian tissue. It accounts for up to 50% of the bodily phospholipid
composition.
It makes up a very high proportion of the outer leaflet of the plasma membrane. Phosphatidylcholine is also the principal
phospholipid circulating in plasma, where it is an integral component of the lipoproteins, especially the HDL.
In addition to its function as a membrane constituent, phosphatidylcholine has a role in signalling via the generation of
diacylglycerols, especially in the nucleus. Discuss DAG PIP3 pathway here!
Phosphatidylcholine is the biosynthetic precursor of sphingomyelin and as such must have some influence on the many
metabolic pathways that constitute the sphingomyelin cycle. It is also a precursor for phosphatidic acid,
lysophosphatidylcholine and platelet-activating factor, each with important signalling functions, and of
phosphatidylserine.

Spliceosome
The introns (non-coding) and exons (coding) of a gene both undergo transcription into pre-mRNA. However, before
translation can occur, a post-transcriptional modification known as ‘splicing’ is required. Splicing removes the non-coding
introns and ligates together the exons. For many eukaryotic introns, splicing is done in a series of reactions which are
catalyzed by the spliceosome, a complex of small nuclear ribonucleoproteins (snRNPs). (Note that some self-splicing
introns known as ribozymes exist.)
The spliceosome is a complex set of machinery consisting of 5 snRNA molecules, each less than 200 nucleotides each in
length, and as many as 200 proteins. The 5 RNA molecules are known as U1, U2, U4, U5 and U6.
The snRNA of the spliceosome have a nucleotide sequence that is homologous to the 5’ ‘splice donor sequence’ end of
the pre-mRNA intron. Introns are identified by ‘branch sites’ in the centre of their sequence. These sequences base pair
with one another to allow for the formation of the spliceosome. Two trans-esterification reactions occur when the
spliceosome is fully formed which result in the excision of the intron through a looping structure.
Splicing offers the advantage of ‘alternative splicing’ producing multiple proteins from the same genetic sequence, but it
also comes with the risk of losing an entire transcript due to an aberrant splicing.
An example of alternative splicing is that of the fibronectin gene. When spliced in different places it can lead to an mRNA
transcript encoding either fibroblasts or hepatocytes.
Evidence for the splicing of the non-coding introns in a genetic sequence is the formation of heteroduplex loops which can
be viewed under an electron microscope. Hybridization between the nucleic acids that are not perfectly complementary
results in the heteroduplex formation. These loops are essentially the introns that were present in the pre-mRNA but not in
the mRNA resulting in their ‘looping’ out and remaining unpaired.

Proline
Proline (abbreviated as Pro or P) is an α-amino acid, one of the twenty DNA-encoded amino acids. It is neutral and non-
polar. Its codons are CCU, CCC, CCA, and CCG. It is not an essential amino acid, which means that the human body can
synthesize it. It is unique among the 20 protein-forming amino acids in that the amine nitrogen is bound to two alkyl
groups, thus making it a secondary amine. Proline is the only cyclic amino acid. The more common L form of proline has
S stereochemistry.
A study done earlier this year showed the important role of proline in plants exposed
to various stress conditions. Besides acting as an excellent osmolyte, proline plays
three major roles during plant stress; as a metal chelator, an antioxidative defense
molecule and a signaling molecule. Proline is optimally detected using a
spectrophotometer at 510–530 nm. Pro is synthesized from Glutamic acid. Proline
residues confer unique structural constraints on peptide chains and markedly influence the susceptibility of proximal
peptide bonds to protease activity.
Proline is often found at the end of α helix or in turns or loops. This is because proline is in a peptide bond where it does
not have a hydrogen on the α amino group. This means it cannot donate a hydrogen bond to stabilize an α helix or a β
sheet leaving a kink in alpha-helices.
Chromatin
In eukaryotes, protein coding genes are composed of a string of alternating introns and exons associated with regulatory
regions of DNA. Chromatin is the mass of genetic material composed of the DNA, histones and non-histone proteins that
can condense to form chromosomes during eukaryotic cell division. The human genome contains 3.2x10^9 DNA
nucleotide pairs, divided between 22 pairs of autosomal chromosomes and two sex chromosomes.
The DNA in Eukaryotes is tightly bound to an equal mass of histones which makes up nucleosomes. Nucleosome is
composed of an octameric core of histone proteins. Nucleosomes are generally spaced approximately 200 nucleotides
apart from one another.
Chromatin exists in two forms in our cells. A) Heterochromatin is tightly compacted and contains genes which are not
being actively expressed. B) Euchromatin is (relatively) more loosely packed and contains genes which are being actively
expressed.
During DNA replication, chromatin undergoes remodelling which allows important proteins and transcription factors to
access the DNA associated with the nucleosome core. It does this by disrupting histones, in particular the H2A:H2B
dimer.
An example of an important heterochromatic sequence is the telomere of chromosomes. (Recall sequence- TTAGGGG, as
many as 1000 copies at the end of chromosomes, telomerase = enzyme, aging and natural shortening which induces cells
to become senescent, cancerous cells take over the enzyme for immortalization.)
When a gene that is normally expressed in euchromatin is experimentally relocated into a region of heterochromatin it
becomes silenced. This shows the importance of gene positioning.
During prophase of mitosis, chromatin fibers become coiled into chromosomes with each chromosome having two
chromatids joined at a centromere.
Certain types of chromatin structure can be inherited….epigenetic inheritance. Just remember chromatin = unwound
chromosomes.

Tyrosine
Tyrosine, an essential, nonpolar, hydrophobic, aromatic amino acid derived from phenylalanine by
hydroxylation at the para-position.
Tyrosine absorbs ultraviolet radiation and hence contributes to the absorbance spectra of proteins.
Tyrosine is a precursor of important neurotransmitters such as epinephrine, norepinephrine and
dopamine.
Tyrosine is encoded by UAU and UAC.
Tyrosine hydrogen bonds make a large contribution to protein stability.
Tyrosine phosphorylation is one of the key covalent modifications that occurs in multicellular
organisms as a result of intercellular communication. The enzymes that carry out this modification are
the protein tyrosine kinases (PTKs), which catalyse the transfer of the phosphate of ATP to tyrosine residues on protein
substrates. Phosphorylation of tyrosine residues modulates enzymatic activity and creates binding sites for the recruitment
of downstream signalling proteins.
Nucleotide excision repair
The inability to repair DNA damage properly can lead to many disorders and enhanced rates of tumor development.
Mammals respond to chromosomal insults by activating a complex damage response pathway that regulates responses
such as cell cycle arrest and apoptosis. NER (Nucleotide Excision Repair) acts on a wide diversity of DNA lesions such as
bulky chemical adducts, DNA intrastrand cross links, and some forms of oxidative damage. However, Pyrimidine Dimers,
caused as by exposure to the sun’s UV, are the most significant type of DNA damage NER must repair.
Pyrimidine dimers prevent DNA polymerases from replicating DNA strands beyond the dimer formation. UV specific
endonucleases can recognise the dimer and cleave the damaged strand both the 3’ and 5’ of it. A short oligonucleotide
sequence containing the dimer is released. The gap created is filled using DNA polymerases and DNA ligases.
NER requires the action of more than 30 proteins in a stepwise manner for the recognition of the damage, opening the
DNA duplex locally around the lesion, dual incision of the damaged DNA strand, gap repair synthesis, and strand ligation.
At least three syndromes are associated with inborn genetic defects in NER: XP (Xeroderma Pigmentosum), CS
(Cockayne Syndrome) and TTD (Trichothiodystrophy), all characterized by exquisite sun sensitivity. XP exhibits a
dramatic >1000-fold incidence of sun-induced skin cancer.

Ubiquitin
Eukaryotic proteins can be modified through their attachment to various small molecules and proteins. Ubiquitin is a
small protein that is extremely well conserved among Eukaryotes. Ubiquitin and ubiquitin-like proteins can be transiently
attached to thousands of different proteins as a monomer or as a poly-ubiquitin chain in a process known as
ubiquitination.
Often when a substrate protein is coupled to a poly-ubiquitin chain, it binds to the 26S proteasome, a large multisubunit
protease complex that degrades the substrate into small peptides and recycles the ubiquitin tag. This happens for
substrates that must be eliminated for proper cell-cycle progression, transcriptional regulation, protein quality control,
signal transduction or circadian rhythms.
Ubiquitylation is also used in non-proteolytic regulatory mechanisms, such as membrane-protein endocytosis and
intracellular trafficking, chromatin-mediated regulation of transcription, DNA repair and the assembly of signalling
complexes.
Diseases which have formed as a result of mis-regulation of the ubiquitin system currently include: many cancers, some
severe types of mental retardation (such as Angelman syndrome), neurodegenerative disorders such as Parkinson's
disease, Huntington's disease and Alzheimer's disease, and type 2 diabetes.

Action potential
An action potential, or nerve impulse, is a series of electrical responses that occur in the cell. With the appropriate
stimulation, the voltage in the dendrite of the neuron will become somewhat less negative. This change in the membrane
potential, called depolarization, will cause the voltage-gated sodium channels to open. Sodium ions will rush in, resulting
in a rapid change in the charge. At the peak of the action potential, that area of the neuron is about 40 mV positive. As the
voltage becomes positive, the sodium channels close, or inactivate, and the voltage-gated potassium channels open. These
potassium channels let potassium ions rush out of the cell, causing the voltage to become negative again. The potassium
channels remain open until the membrane potential becomes at least as negative as the resting potential. In many cases,
the membrane potential becomes even more negative than the resting potential for a brief period; this is called
hyperpolarization. An action potential typically lasts a few milliseconds.
Propagation of action potential: When the sodium channels are opened, sodium ions rush in; once inside they cause
nearby regions of the neuron to become depolarized by moving laterally through
the axon. This, in turn, causes the opening of more voltage-gated sodium
channels in those regions. Thus, the sodium channel activation moves in a wave-
like fashion: the action potential is propagated down the length of the neuron,
from its input source at the dendrites, to the cell body, and then down the axon to
the synaptic terminals. After opening, the sodium channels become inactivated
as the potential becomes more positive, and they cannot open again until they are
"reset" by hyperpolarization at the end of an action potential. This brief period of
sodium channel inactivation, called a refractory period, prevents bidirectional
propagation of the action potential, constraining it to go in only one direction.

Drug Bioavailability
Drug bioavailability refers to the fraction of a dose of drug that is absorbed from
its site of administration and reaches, in an unchanged form, the systemic
circulation. The drug itself, its route of administration and its galenic formulation (<- deals with the principles of
preparing and compounding medicines in order to optimize their absorption), determine the amount of administered dose
absorbed into the circulation. Some factors which influence bioavailability are:
 Physical properties of the drug (hydrophobicity, pKa, solubility)
 The drug formulation (manufacturing methods for immediate release, delayed release, extended release, sustained
release, etc.)
 Whether the drug is administered in a fed or fasted state
 Gastric emptying rate
 Interactions with other drugs (e.g., antacids, alcohol, nicotine)
 Interactions with other foods (e.g., grapefruit juice, cranberry juice, certain vegetables)
 Transporters concentration: Substrate of efflux transporters (e.g. P-glycoprotein)
 Health of the GI tract of patient
 Age: In general, drugs are metabolized more slowly in fetal, neonatal, and geriatric populations
 Disease state E.g., hepatic insufficiency, poor renal function
 Surface time available for absorption
Bioavailability of most small molecular weight drugs administered i.m. or s.c. are perfusion rate-limited. Large molecules
administered i.m or s.c. enter the blood in part via the lymphatic pathway.

Urea cycle
Urea is the major end product of nitrogen metabolism in humans and mammals. Most of our nitrogenous waste comes
from the deamination of amino acids. Ammonia, the product of deamination, is toxic in even small amounts and must be
removed from the body. The urea cycle describes the conversion reactions of ammonia into urea. Since these reactions
occur in the liver, the urea is then transported to the kidneys where it is
excreted. The overall urea formation reaction is:
2 Ammonia + carbon dioxide + 3ATP ---> urea + water + 3 ADP
One turn of the cycle:
•Consumes 2 molecules of ammonia
•Consumes 1 molecule of carbon dioxide
•Creates 1 molecule of urea ((NH2)2CO
•Regenerates a molecule of ornithine for another turn.
Urea is routinely measured in the blood as: Blood Urea Nitrogen (BUN).
BUN levels may be elevated (a condition called uremia) in both acute and
chronic renal (kidney) failure. In severe cases, hemodialysis is used to
remove the soluble urea and other waste products from the blood. Waste
products diffuse through the dialyzing membrane because their
concentration is lower in the dialyzing solution.

Oestrogen Receptor
Background: Oestrogens are best known for their effects upon the female reproductive system, where they play a major
role in ovulation, implantation, pregnancy maintenance, childbirth and lactation. However, these steroid hormones are
also essential for sperm production in males, and have important physiological functions in the cardiovascular system,
immune system, central nervous system, and in bone, where they support several diverse physiological processes such as
cardiovascular protection, the humoral immune response, neuroprotection and bone remodelling.
The ER: The biological actions of oestrogens are only found in cells expressing oestrogen receptors. Oestrogen receptors
(ERs) act as transcription factors, either activating or inhibiting the expression of a wide array of genes. Cells can respond
to oestrogen in different, often opposing ways, because of the presence of two functionally distinct oestrogen receptors
and their ability to interact with a number of different cofactors and signalling proteins. There are two receptors that can
bind to oestrogen, the oestrogen receptor alpha (ERa) and the oestrogen receptor beta (ERb). These receptors are
functionally distinct, having different tissue distributions and different ligand activation, and as such play different roles in
gene activation. Both ERa and ERb interact with the same oestrogen ligand, oestradiol-17b, but they behave differently,
sometimes even causing opposing effects. For example, ligand-bound ERa was found to activate transcription, while
ligand-bound ERb inhibited transcription from the AP1 site.
The ER and breast cancer: Oestrogen receptors are required for oestrogen stimulated growth and proliferation of breast
cancer cells. They are found to be upregulated in some degree in approximately 75% of breast tumours. Endocrine
treatments are designed to antagonise the effects of oestrogen. For example, antioestrogens such as tamoxifen
competitively block the binding of oestrogen to its receptors and thus antagonise transcriptional activation of genes
required for tumour growth.
Breast cancer tumors that are ER/PR-positive are 60% likely to respond to endocrine therapy. Tumors that are ER/PR
negative are only 5% to 10% likely to respond to endocrine therapy.
One major way of defining the type of breast cancer is whether or not it is:
 Endocrine receptor (estrogen or progesterone receptor) positive
 HER2 positive
 Triple negative, not positive to receptors for estrogen, progesterone, or HER2
 Triple positive, positive for estrogen receptors, progesterone receptors and HER2

Cholesterol
Cholesterol is a steroid which modulates the fluidity of animal cell membranes by increasing the cohesion and packing of
membrane lipids. In the plasma membrane cholesterol typically accounts for 20–25% of the lipid molecules. Cholesterol
is also abundant in the Golgi complex, with enrichment towards the trans-Golgi compartments.
Cholesterol is the precursor of many steroid hormones such as progesterone, testosterone, and cortisol. All 27 carbon
atoms of cholesterol are derived from acetyl CoA in a three-stage synthetic process…
1. Stage one is the synthesis of Isopentenyl pyrophosphate, the key building block of cholesterol.
2. Stage two is the condensation of six molecules of Isopentenyl pyrophosphate to form squalene.
3. In Stage three, squalene cyclizes and the tetracyclic product is subsequently converted into cholesterol.
In humans, the body cholesterol is derived from two
sources — diet and de novo synthesis at a ratio of 30:70.
The degree to which serum cholesterol is increased by
dietary cholesterol depends upon whether the individual's
cholesterol synthesis is stimulated or down-regulated by
such an increased intake, and the extent to which each of
these phenomena occurs varies from person to person.
The de novo synthesized cholesterol leaves the ER
rapidly, mostly by non-vesicular mechanisms that bypass
ER–Golgi membrane transport, thereby helping to
maintain low ER sterol content. The biosynthetic sterols, including cholesterol and its precursors are rapidly targeted
to the plasma membrane and become available to extracellular acceptors.

Protein phosphorylation
It is now generally accepted that protein phosphorylation/dephosphorylation has a role in the regulation of essentially all
cellular functions. This is because almost every known signalling pathway involves a protein kinase or protein
phosphatase.
The first signalling cascade in which protein phosphorylation/dephosphorylation was found to be involved was elucidated
30–40 years ago.
Proteins can be reversibly phosphorylated on their tyrosine, serine and threonine residues.
G-protein-coupled receptors generate signals that promote gene transcription through the activation of RTKs and the
mitogen-activated protein kinase (MAPK) cascade. RTKs activate themselves through autophosphorylation upon ligand
binding. This phosphorylation event provides phosphotyrosine docking sites for effectors, adaptors and scaffold proteins.
These then perpetrate the signal in various ways e.g. through the induction of secondary messengers such as cAMP.
The initial phosphorylation events of RTKs generally involve only the phosphorylation of tyrosine residues, but the
amplification processes mostly involve serine/threonine phosphorylations. Protein tyrosine phosphatases (PTPs) are
highly conserved phosphatases that regulate the tyrosine phosphorylation (ie PTPs have the ability to remove the
phosphate group).
The availability of numerous X-ray crystal structures of RTKs and PTPs, along with their inhibitors, has provided the
opportunity for the structure-based design of effective inhibitors having the potential for the treatment of various disorders
such as cancer.

Eukaryotic Ribosome
All ribosomes are composed of two subunits, both of which are built from RNA and protein. In contrast to their bacterial
counterparts, eukaryotic ribosomes are much larger and more complex, containing additional rRNA in the form of so-
called expansion segments (ES).
The eukaryotic ribosome is a complex assembly of four ribosomal RNAs (rRNA) with about 80 ribosomal proteins (rps).
The preeminent role of the ribosome is to translate messenger
RNA into polypeptides. The small ribosomal subunit in yeast is composed of an 18S rRNA and 32 rps, whereas the 60S
subunit is composed of 3 rRNAs and 46 rps. The rRNA ‘‘core’’ of the 80S ribosome is homologous to that of the bacterial
70S ribosome.
The catalytic core of the ribosome—where aminoacylated-tRNA is decoded, peptide bonds are formed, and tRNAs are
translocated from defined binding sites of the ribosome to the next— is highly conserved in the ribosomes of all species of
life. Cryo-EM is currently being used to decipher the structure of other eukaryotic ribosomes.
This entire process is carried out by the ribosome which acts as a loading dock for tRNA molecules and moves along the
mRNA strand, beginning at an AUG start codon near the 5’ end. It recognises the start sequence by use of the Shine-
Dalgarno sequence of prokaryotic mRNA or the Kozak box in eukaryotes. These are short sequences located just
upstream from the start codon. The rRNA forms a highly structured pocket that, through hydrogen bonding, correctly
orientates the tRNA and mRNA allowing for them to covalently join together in an accelerated fashion. The ribosome
contains three RNA binding sites, A, P and E. mRNA binds to the P site of the ribosome first which which binds peptidyl-
tRNA i.e. tRNA bound to the peptide being synthesised. The A site binds aminoacyl-tRNA and the E site binds a free
tRNA before it exits the ribosome. The ribosome is universally conserved in all living things. Prokaryotic and eukaryotic
ribosomes only really differ in the number and size of their rRNA and protein components. They have almost the same
structure and are functionally similar. It is possible for some drugs to exploit the differences between bacterial and
eukaryotic ribosomes, interfering preferentially with the function of bacterial ribosomes. Because of this humans can take
high doses of these drugs without undue toxicity. Many antibiotics function by lodging in the pocket of bacterial
ribosomes and blocking the important steps necessary for protein synthesis.

Caspase 3
Caspase 3 is a protein in the cysteine aspartic acid protease family. Caspases are important mediators of apoptosis. They
are synthesized as inactive precursors (zymogens) that then must be cleaved at conserved aspartic acid residues to become
active. Caspase 3 is made up of a large subunit and a small subunit. Caspase 3 is 32kDa in size. (It is cleaved into 17kDa
and 12 kDa subunits). Caspase 3 can be activated by extrinsic (death ligand) or intrinsic (mitochondrial) pathways. It goes
on to further propagate caspase signals, activating caspase 2 and 6. It is also thought to be involved in a positive feedback
loop that activates caspase 9 in the apoptosome. Alternatively, it may also be the case that caspase 3 is cleaving caspase 9
at a position that affects the binding of caspase9 and it’s inhibitor XIAP.
Caspase 3 functions directly as a mediator of apoptosis. An example of how caspase 3 is involved in apoptosis is the
cleavage of ICAD-CAD (Caspase Activated DNAase) which then allows CAD to go on and fragment the DNA. It is a
substrate of granzyme B which is a vital protein in apoptosis (along with perforin). There has been a correlation with
caspase 3 activation and neurodegenerative diseases such as Alzheimers, Parkinsons and Hungtingtons.

Disulphide bonds in protein

Disulfide bridges form from the coupling of two thiols:

R-SH + R-SH —> R-S-S-R +2H + 2 e-

In some proteins the linear polypeptides chains are cross-linked. Disulfide bonds are the most common type of cross links
found in proteins. The disulfide bond is weaker than the C-C and C-H bond and as a result is more likely to be broken.
They are formed as a post translational modification (oxidative reaction) in the ER. Within proteins, disulfide bonds are
formed by two cysteine residues to make a cystine. Internal disulfide bridges (x-cys-cys-) are common in proteins
excreted from the cell or those exposed to extracellular conditions. They affect the structural stability of the protein acting
as atomic staples that stabilise the proteins conformation. They do this by bridging the two parts of the protein together
and by being the centre in which hydrophobic interactions are made by other amino acids in the protein. They also work
by promoting resistance to proteases and increase the thermodynamic stability of the protein. An example of the
importance of disulfide bonds in protein is insulin. Insulin is composed of an A and B chain which are linked by a
disulphide bond. Disulfide bonds can be cleaved in proteins by assaying the reducing agent marcaptoethanol.
Mercapthethanol can be added to a protein sample before SDS page to break the disulfide bonds and denature the protein.

IgA
IgA is an antibody that is found mainly in the mucus lined areas of the body such as the the GI tract or respiratory
epithlium. IgA is found as either a polymer or a monomer. Polymeric IgA is composed of two IgA monomers attached by
a J chain (which is a polypeptide that is cysteine rich). The only other antibody that has a J chain is pentameric IgM. Both
IgA and IgM are secreted in the human body. There are two subtypes of IgA:
IgA1 – monomeric found mainly in serum, activate alternative complement pathway.

IgA2 – polymeric form mainly found in secretions.

The mean level of IgA in the serum is 1.2 mg/ml, IgA is found in saliva, tears and colostrum. IgA mainly functions
through neutralization and by transporting across the epithelium. Polymeric IgA is transported through epithelial cells at
the base of the crypt. It does so by binding to the polyIG receptor on the basolateral surface of the cell. It is then
transported through the cell wall via endocytosis to the luminal surface of the epithelial surface. Once there, it acts
through neutralization of the bacterial antigens on the bacterial membrane. IgA also functions in bacterial lysis. Individual
B cells are committed to secreting either monomeric or polymeric IgA. IgA deficiance causes 2.5% of Coeliac disease
diagnosis.

L-SELECTIN
Also called CD62L is an adhesion molecule found on lymphocytes. It belongs to the selectin family of proteins that
recognise sialylated carbohydrates. It is the smallest of the selectin molecules at 74-100 kDa. It is a type I membrane
protein with a C type lectin at its amino terminus, an EGF domain, two short consensus repeats, a transmembrane domain,
and a short cytoplasmic tail. L-selectin is important in leukocyte homing to secondary lymphoid tissues and for adhesion
to endothelial cells. It is therefore a key player in inflammation. Upon T cell activation L-selectin is shed from the surface
of leukocytes. The reduced expression of L-selectin allows leukocytes to leave the bloodstream and come in contact with
endothelial cells. Transmigration through endothelial cells allows the leukocyte to encounter the antigen. The ligands of
L-selectin found on endothelial cells are: GlyCAM-1, CD34, sgp200, PNaD and MAd -CAM-1. L-selectin is involved in
chronic inflammation diseases such as Crohn’s disease making it a good drug target for intervention in such diseases.
Examples of drugs that target L-selectin are EL-246.

Receptor mediated endocytosis

RME is also known as clathrin dependant endocytosis. Endocytosis is the process by which the plasma membrane
invaginates and forms a vesicle due to the binding of a ligand to its receptor. The vesicle containing both the receptor and
ligand is coated in clatherin. Clatherin is a protein that forms a triskelion shape. When separate triskelions interact they
form a polyhedral lattice around the vesicle. The vesicles clatheirn coat is shed when the vesicle is formed. Examples of
receptor mediated endocytosis are the transport of cholesterol (LDL), iron and EGF.
In the case of cholesterol, cholestrol is transported in the blood in the form of cholesteryl esters that are LDL. When the
cell needs cholesterol it synthesizes LDL receptors that are then embedded in the membrane. The receptors are associated
with clatherin. When the LDL binds to its receptor, it is then internalised within the coated vesicle. The clatherin coat is
shed and the contents of the vessicle go into the ‘early endosome’ which is at the periphery of the cell. The low pH of the
endosome causes the LDL and its receptor to dissociate with the LDL going from the late endosome to the lysosome
where they are hydrolyzed from cholesteryl esters to free cholesterol which goes on to become a constituent of the plasma
membrane.
 The low pH of the endosome causes the LDL and it’s receptor to
dissociate with the LDL going from the late endosome to the
lysosome where they are hydrolyzed from cholesteryl esters to
free cholesterol which goes on to become a constituent of the
plasma membrane.If there is too much cholesterol in the
membrane the cell can turn off synthesis of the LDL receptors or
of proteins involved in cholesterol synthesis. Mutations in genes
coding for the LDL receptor can cause artheriosclerosis where the
cholesterol is not taken into the cell but instead forms plaques in
the blood vessels (hypercholesteremia).

Histone acetyl transferase

Histone acetyl transferases (HATs) are a type of enzyme that add acetyl groups to amino acids found on histones in
chromatin. The source of the acetyl groups is acetyl-CoA. Histones are proteins made up of H2A, H2B, H3 and H4 that
form an octamer in which the DNA is wrapped around. Histones have histone tails which are globular proteins that have
conserved residues and undergo modifications.
Histone acetyltransferases act primarily on Lysine (K) residues. The addition of chemical groups to the residues changes
their charge and can affect the interactions between DNA and the histone and histone/histone interactions. The
modification to the protein can be recognised by other proteins and can activate transcription. In the case of HAT, the
process is reversed by histone deacetylases (HDAC). HAT’s a diverse range of enzymes that can also act on substrates
other than histones. They function in DNA stability and DNA repair and as transcription factors (FAT- factor
acetyltransfeases). Characterisation of HAT is done by their catalytic domains. Two of the major HAT family proteins are
GNAT and MYST.

The peptide bond

Proteins are linear polymers formed by linking the α-carboxyl group of one amino acid to the α-amino group of another
amino acid. This covalent bond is called a peptide bond.

 Biosynthesis of the peptide bond requires

input of free energy.
 Distance between C-N bond is 1.32Å.
 The peptide bond is planar.
 Resonance structure gives the peptide bond
double bond character making it
thermodynamically stable.
 The formation of a dipeptide bond causes the
loss of water.
Peptide bond synthesis is an enzymatically controlled process that occurs in the ribosome with an mRNA template.
Peptide bonds can be reversed in a hydrolysis reaction which is also enzymatically regulated. A series of amino acids
joined by a peptide bond form a polypeptide chain, Polypeptide chains have polarity. The amino group by convention is
said to be the start of the polypeptide. Almost all peptide bonds in proteins are in the cis-conformation.

Prions
Prions are misfolded proteins that are often associated with neurodegenerative diseases such as BSE (bovine spongiform
encephalitis), creutzfeltd-jakob disease in humans and scrapie in sheep.These diseases are thought to be transmitted purely
by the misfolded proteins. The normal cellular form of a protein, PrPc, is converted to the infectious form PrPsc by means
of a post-translational modification which increases the number of β-sheets. This changes the tertiary structure of the
protein causing aggregates of the PrPsc to form. (These fibrous aggregates are thought to act as nuclei in which other PrP
molecules attach.) The aggregates go on to form insoluble amyloid fibres. Because prion related diseases spread by
misfolded proteins causing other proteins to misfold, it is easily transferrable.
Examples of outbreaks are the outbreak of BSE in the UK in the 1990’s caused by cattle being fed animal feed containing
components of other dead cattle. Another example is the transmission of kuru in Papua New guinea by cannibilistic
funeral rituals. Symptoms of prion diseases in humans include dementia, memory loss, personality changes, hallucinations
and ataxia (balance dysfunction). The symptoms of the disease are caused by progressive neuronal death that will
eventually be fatal. There has been links made between Alzheimer’s disease and CJD as both come as a result of amyloid
plaques.

Hypercholesterolemia
Familial hypercholesterolemia is a genetic disorder where the Low Density Lipoproteins (LDL) receptor is absent
resulting in increased levels of cholesterol in blood vessels. Cholesterol is transported in the blood in the form of LDLs
that are taken up by LDL receptors via receptor mediated endocytosis. The levels of cholesterol and LDL in the blood are
markedly elevated in hypercholesterolemia, which is the result of a mutation at single autosomal locus. Homozygous
cholesterol plasma levels are typically 680 mg/dl, compared with 300mg/ml in heterozygotes. (The optimum cholesterol
level is typically under 200mg/dl.) The elevated cholesterol levels result in deposits in various tissues called xanthomas
which are prominent in skin and tendons. The disorder also affects the immune system as oxidised forms of LDL called
oxLDL are taken up by macrophages, which then become engorged and form foam cells. Foam cells become trapped in
the walls of blood vessels, aiding the formation of arteriosclerotic plaques.
The only way to treat homozygous familial hypercholesterolemia is a liver transplant. Heterozygotes are treated in two
ways 1) The inhibition of reabsorption of bile salts from the intestine (cholestyramine) 2) Blocking de novo cholesterol
synthesis (statins). Both these treatments are attempting to increase LDL receptor numbers in the cell by cutting off
cholesterol supply.

Topoisomerase
Topoisomerase is a class of enzyme that introduces and eliminates supercoils in DNA. They come in two forms type 1 and
type 2 topoisomerases. Type 1 topoisomerases cleave one strand of the DNA. They have 4 domains which are arranged in
a cavity of 20 Å that is just big enough for ds-DNA to fit into. The cavity also contains a Tyr 723 residue which acts as a
nucleophile to cleave the DNA backbone. The mechanism for type one topoisomerase is as follows:
1) The DNA molecule binds inside the topoisomerase cavity. The OH group of the Tyr attacks the phosphate group of the
DNA backbone to form a phosphodiester linkage between the enzyme and the DNA cleaving the DNA and releasing a
free 5’OH group
2) The DNA rotates around the remaining strand causing the supercoil to unwind
3) The free 5’OH group of the DNA attacks the phosphotyrosine residue to reseal the backbone and release the tyrosine
4) The enzyme is then able to disassociate with the DNA.
Type II topoisomerases utilize free energy from the hydrolysis of ATP to add negative supercolis to the DNA. Their
mechanism of action is:
1) Topoisomerase II binds to a DNA duplex termed the ‘G segment’.
2) The binding of ATP to the N terminal domain brings two of the domains together leading to cleavage of both strand of
the G segment.
3) Another segment of DNA called the ‘T segment’ binds to the topoisomerase. The action of binding the T segment
causes cleavage in two strand of the G segment.
4) The T segment then moves through the enzyme and the G segment unlike with type one does not rotate instead the
strength of the bond introduces negative supercoils to the DNA. The DNA is then resealed and released.
The bacterial topoisomerase II (DNA gyrase) is the target of several antibiotics such as novobiocin that blocks ATP
binding to the gyrase.

Isoelectric point
Isoelectric point or pI is the pH at which a molecule has no net electrical charge. To calculate the pI of an uncharged
amino acid you use the formula:
Below the pI the molecule can be positively charged and above it the molecule is negatively
charged. The pI of a molecule is utilized in protein separation techniques such as isoelectric
focusing. Isoelectric focusing is an electrophoretic method by which proteins are separated
based on their relative pI’s. The net charge of a protein is the sum of positive and negative charges on its side chains: If
the number of acid groups on the protein exceeds the number of bases then the Pi of the protein will be low and it can be
termed acidic. If the basic group exceeds the acidic group the pI is high and the protein is said to be basic. Protein
isoelectric points tend to fall between pH 4-7. Proteins that are positively charged (in solution) at pH values below their
isoelectric point and negatively charged at values above their Pi. So at pH values below its pI the protein will migrate
towards the cathode during electrophoresis. At pH values above its pI the protein will migrate to the anode.

Proteins in a solution at their pI will not move in the field. At pH values lower than the pI, the protein is positively
charged and moves towards the cathode. At values above the pI the protein is negatively charged and will move towards
the anode.

Autophagy
Autophagy is a catabolic process involving the
degradation of a cell’s worn out organelles by its
lysosomes. Autophagy begins with the enclosure of an
organelle by a double membrane, of unknown origin,
creating the autophagosome. The autophagosome then
fuses with a lysosome or a late endosome for its
contents degradation. This process is highly regulated.
A target must be selectively marked for lysosomal
degradation. Autophagy helps restructure differentiating cells by disposing of no longer needed organelles and as a
defence mechanism against invading viruses/bacteria.

ELISA
Enzyme-Linked Immunosorbent Assays combine the use of antibodies and colorimetric changes to detect the presence of
a certain substance, usually an antigen, in a liquid sample. ELISAs are often used to verify the identity of a protein and
also to determine concentrations of proteins in conjunction with mass spectroscopy. There are different types of ELISA
such as indirect and competitive but the most commonly used type is the sandwich ELISA. In this method a 96 well plate
is coated with the sample. The plate is covered in a blocking serum to prevent non-specific binding and any unbound
serum is washed away. Next a primary antibody is added which binds to the antigen of interest. Again unbound antibody
is washed off. A secondary antibody is then added. This antibody is designed so that it binds to the nonspecific Fc region
of the primary antibody. It is usually also biotinylated which allows it to be detected and bound by streptavidin-HRP
(horse radish peroxidise) which allows for detection. Finally a substrate is added to the wells and colour is produced by
the enzyme activity of peroxidise. This allows for quantification of the bound antibody (and therefore the antigen) in the
wells. ELISA is widely used as a reliable screening test for diseases such as malaria, hepatitis B and HIV.
Lysine
Lysine (Lys, K) is an α-amino acid. It is an essential amino acid meaning that although it is synthesised in plants and
bacteria animals are unable to produce lysine and so it must be ingested as lysine or lysine-containing proteins. In plants
and bacteria it is synthesised from aspartic acid. In mammals, lysine is metabolised to produce acetyl-coA an important
molecule which is used in many biochemical reactions and is vital to metabolism. Lysine can be industrially
manufactured. It is a necessary building block for all protein in the body and is important for calcium absorption, building
muscle protein, recovery after injury and production of hormones, enzymes and antibodies. Modification of lysine of
lysine residues by acetylation, ubiquitination etc. can modify the function of the protein which the lysine is a part of.
Lysine has been identified as a possible treatment for a number of diseases and conditions including HSV (Herpes
Simplex Virus), anxiety disorders and cancer. In cancer lysine methylation of p53 has been shown to stabilize the
transcription factor and regulate the expression of its target genes depending on the site of methylation. As well as this
many animal studies have shown that lysine deficiencies can lead to immunodeficiency.

IL-10
Interleukin 10 (IL-10) is an anti-inflammatory cytokine also known as human cytokine synthesis inhibitory factor (CSIF).
It is a 178aa long homodimer protein and contains intrachain disulfide bonds which are essential for its function. It is
primarily produced by monocytes and on a lesser level by some lymphocytes such as T-regulatory cells and Th2 helper
cells. IL-10 is released by cytotoxic T-cells to inhibit the action of natural killer cells during the immune response to viral
infection. IL-10 has both immunoregulatory and anti-inflammatory effects. It is responsible for the downregulation of Th1
cytokine expression, MHC class II antigens, and co-stimulatory molecules on macrophages. It enhances B cell survival,
proliferation, and antibody production and can block NF-κB activity as well as being involved in the regulation of the
JAK/STAT signalling pathway. IL-10 is capable of inhibiting synthesis of pro-inflammatory cytokines including IFN-γ,
IL-2, IL-3, TNF-α and GM-CSF made by macrophages and regulatory T cells. It can also suppress the ability of antigen
presenting cells which drive immune responses. Mouse studies have shown that mast cells can produce IL-10 which
counteracts the inflammatory response in an allergic reaction.

Biotin
Biotin (Vitamin H, coenzyme R) is a water soluble B-vitamin. It consists of two tetrahydrothiophene rings fused together
with a valeric acid side chain. Biotin is essential for cell growth, the production of fatty acids and the metabolism of fats
and amino acids. It also plays an important role in the citric acid cycle, the process by which biochemical energy is
produced during anaerobic respiration. Biotin is produced by intestinal bacteria and as it tends to be produced in excess of
the body’s daily requirements, biotin deficiencies are rare. Biotin also acts as a cofactor for carbon dioxide transfer in
several carboxylase enzymes such as pyruvate carboxylase. Biotin is widely used in biotechnology to conjugate proteins
for biochemical assays in a process called biotinylation. Due to its high affinity for avidin and streptavidin, this interaction
can be exploited to isolate biotinylated proteins in processes such as ELISAs. Biotin has also been used to aid diabetics
with either type I or type II diabetes as supplementation with biotin can improve blood sugar control and help lower
fasting blood glucose levels.

Telomeres
Telomeres are guanine-rich regions of repetitive nucleotide sequences which are found at the ends of chromosomes in
eukaryotic DNA. Their lengths vary greatly between different species ranging from a few hundred base pairs in yeast to
many kilobases in humans. Telomeres function to protect the ends of chromosomes from degradation or from annealing to
adjacent chromosomes and, as most prokaryotic DNA is circular, they are not necessary for the majority of prokaryotes.
Chromosomes are replicated during cell division however the enzymes responsible for this are unable to replicate the
entire chromosome and as a result without the presence of telomeres at the ends chromosome replication would be
incomplete and important genetic information would be lost. Instead the telomere is lost and subsequently replenished by
telomerases. With each cell division the telomere length is shortened until eventually the chromosome can no longer by
fully replicated the cell dies (senescence). This limited division of cells is known as the Hayflick limit and occurs
naturally with the aging process. Telomerase is only present in small numbers in somatic cells and is generally only
expressed in high levels in rapidly proliferating cells such as stem cells. It plays an important role in tumour formation and
the progression of cancer where its expression up-regulated. Because of this telomerase acts not only as a successful
biomarker but is also viewed as a possible target for anti-cancer drugs.

Phosphatases
A protein phosphatase catalyses dephosphorylation (phosphate removal). They remove a phosphate group from its
substrate by hydrolysing phosphoric acid monoesters into a phosphate ion and a molecule with a free hydroxyl group.
There are many different protein phospahatases, some very specific while others act on a wide range of proteins and are
targeted to specific substrates by regulatory subunits. The constant phosphorylation and dephosphorylation of proteins
allows them to switch rapidly from one state to another e.g. active to inactive, a process powered by ATP. The human
genome encodes about 150 protein phosphatases. Many signalling proteins, such as G-proteins, are controlled by the
combined actions of kinases and phosphatases.

The pentose phosphate pathway

The pentose phosphate pathway (also known as the phosphoglucaonate pathway or the hexose monophosphate shunt) is
the pathway by which NADPH and pentoses, 5-carbon sugars, are produced. The pathway is an alternative to glycolysis
but is anabolic rather than catabolic. For most organisms the process occurs in the cytosol. It occurs mostly in the plastids
of plants. In humans it is most active in the liver, mammary glands and adrenal cortex. The pathway occurs in two distinct
phases. In the oxidative phase two molecules of NADP+ are reduced to NADPH using the energy provided by the coupled
reaction in which glucose-6-phosphate is converted into ribulose-5-phosphate.The reaction is as follows:
Glucose 6-phosphate + 2 NADP+ + H2O → ribulose 5-phosphate + 2 NADPH + 2 H+ + CO2
The NADPH produced is used in the cell for reductive biosynthetic reactions such as fatty acid synthesis and for
prevention of oxidative stress. In the non-oxidative phase the 5-carbon sugars are synthesized. The pentose which is
generated, ribose-5-phosphate, is used in the production of nucleotides and nucleic acids. A four-carbon sugar, erythrose-
4-phosphate is also produced as an intermediate in this pathway and is used in the synthesis of aromatic amino acids
which in turn are precursors for many other biosynthetic pathways. The pentose phosphate pathway is regulated by the
enzyme glucose-6-phosphate dehydrogenase which is allosterically stimulated by NADP+ for the production of NADPH.
In the event of a deficiency in this enzyme, which is hereditary, individuals may exhibit nonimmune haemolytic anaemia
(the insufficient breakdown of red blood cells) in response to a number of causes, most commonly infection or exposure
to certain medications. Deficiencies in the level of activity of G6PDH (but not its function) have been linked to a
resistance to malaria. This is possibly due to a weakening of the red cell membrane meaning it is unable to sustain the
parasitic life cycle long enough for productive growth.

Ubiquinone
Ubiquinone, also known as coenzyme Q10, is a vitamin-like molecule present in most eukaryotic sells, primarily in the
inner membrane of the mitochondria as its primary role in the cell is to generate energy. It is a component of the electron
transport chain and participates in aerobic cellular respiration to produce energy in the form of ATP. As 90% of the bodies
energy is produced in this way, the organs with the highest energy requirements – such as the heart, liver and kidneys –
have the highest levels of coenzyme Q10. There are three redox states of coenzyme Q10: fully oxidised (ubiquinone),
semiquinone (ubisemiquinone) and fully reduced (ubiquinol). Its ability to exist in both oxidised and reduced forms
allows it to alters its functions as needed for participation in the electron transport chain or for antioxidant purposes. Since
coQ10 is fat soluble it is mobile in membranes and therefore plays a unique role in the electron transport chain,
functioning as an electron carrier from enzyme complex I to enzyme complex II and enzyme complex III. This transport
of electrons results in the pumping of protons across the membrane creating a gradient used by ATP synthase in the
production of ATP. CoQ10 is an energy carrier ands is constantly being oxidised and reduced as it gives up and accepts
electrons respectively. In its reduced form it hold electrons loosely and will readily give up one or both and therefore act
as an antioxidant. Absorption of ubiquinone in the body is similar to that of lipids. It involves secretion of pancreatic
enzymes and bile into the small intestine. These facilitate emulsification and micelle formation which are necessary for
absorption of lipophilic substances. Food intake increases secretion of bile and therefore greatly enhances absorption of
coQ10. Serum levels of coQ10 are lower in the fasting condition. A deficiency in coQ10 may be caused, for example, by a
mutation in genes involved in biosynthesis. CoQ10 deficiency can result in fatigue, muscle weakness, seizures and kidney
failure. There has been evidence of coQ10 deficiencies in heart failure and migraines and research is being done into the
use of coQ10 to relieve some of the side-effects of cancer treatments.

Purines and Pyrimidines

Purines and pyrimidines make up the two groups of nucleotide bases, the building blocks of DNA and RNA. They also
function as a source of energy for cells and are essential for the production of proteins and starch, regulation of enzymes
and cell signalling. In DNA purines and pyrimidines form hydrogen bonds in a process called complimentary base
pairing. Purines adenine and guanine pair with pyrimidines thymine and cytosine, respectively. In RNA adenine is
complemented by the pyriminidine derivative uracil instead of thymine. There are many other naturally occurring purines
which act as significant components of other biomolecules such as ATP, GTP, NADH and cyclic AMP. Examples of
these include xanthine which is found in most human body tissues and fluids in other organisms. Purines may also
function as neurotransmitters acting on purinergic receptors for example adenosine acts on adenosine receptors and
regulates the effects of other neurotransmitters such as dopamine and glutamate. The basic structure of a purine has four
nitrogen atoms and is made up of two carbon rings, a pyrimidine ring fused to an imidazole ring.
A pyrimidine consists of only one carbon ring and has two nitrogen atoms. Other than the three major pyrimidine bases
mentioned, some minor pyrimidine bases can also occur in nucleic acids. They are usually methylated versions of major
ones and are thought to have regulatory fuctions. Many organisms have metabolic pathways to both synthesise and
breakdown purines and pyrimidines. Defects in enzymes that control purine production and breakdown can severely alter
a cell’s DNA sequences, which may explain why people who carry certain genetic variants of purine metabolic enzymes
have a higher risk for some types of cancer. Modulation of pyrimidine metabolism has been shown to have some
therapeutic use for example pyrimidine synthesis inhibitors are used in rheumatoid and psoriatic arthritis to slow
progression of the disease and treat symptoms of inflammation.

Phosphotidylserine
Phosphatidylserine is a phospholipid component found on the inner leaflet of membranes where it is kept by the enzyme
flippase. In the event of apoptosis, it is no longer restricted to the cytosolic side of the membrane and is exposed on the
surface of the cell where it helps signal to neighbouring cells and macrophages to phagocytose the dying cell. In addition
to this it also blocks the inflammation often associated with phagocytosis as the phosphatidylserine-dependent engulfment
of apoptotic cells inhibits the production of pro-inflammatory cytokines by the phagocytic cell. Phosphatidylserine on the
outer layer of the membrane can be visualised by use of labelled Annexin V protein which binds to the phospholipid
making it a useful marker for apoptotic cells. One is application for this is for the distinction between malignant and
benign tumours which have high and low rates of apoptosis respectively. Phosphatidylserine is a diacylglyceride and
contains a diglyceride, a phosphate group and the organic molecule serine. Like all phospholipids phosphatidylserine is
amphipathic and consists of a hydrophilic head group, containing the negatively charged phosphate group, and a
hydrophobic tail group made up of the long fatty acid hydrocarbon chains. Phosphatidylserine is synthesised on the
endoplasmic reticulum membrane facing the cytosol which contains the proteins responsible for the synthesis and
allocation of the phospholipid. Because fatty acids are not soluble they are brought from their site of synthesis to the ER
by a fatty acid binding protein in the cytosol. Once at the ER membrane and after activation with coA, acetyl tranferases
attach two fatty acids to glycerol phosphate to produce phosphatidic acid which is sufficiently water-insoluble to remain
in the bilayer. The serine head group is added and a vesicle buds of from the ER membrane containing the phospholipids
to be transported to the membrane. Phosphatidylserine plays a part in the activation of protein kinase C which, once
activated, phosphorylates different target proteins depending on the cell type.

Protein kinase A
Protein kinase A (PKA) is also known as c-AMP dependent protein kinase as it refers to a family of enzymes whose
activity is dependent on cellular levels of cyclic-AMP. PKAs can phosphorylates serine and threonine residues on specific
cell-dependent target proteins and therefore alter the activity of that protein. Many of these target proteins are also protein
kinases and this leads to a phosphorylation cascade whereby one protein kinase, activated by phosphorylation,
phosphorylates the next protein kinase in the sequence, and so on, relaying the signal onward and, in the process,
amplifying it and sometimes spreading it to other signalling pathways. PKA phosphorylates proteins which have the motif
Arginine-Arginine-X-Serine exposed, causing them to become activated or deactivated. The target protein is dependent on
the cell type which the PKA is present and so the effects of PKA activation vary between cell types. For example, in
adipocytes, the β-adrenergic receptor binds an epinephrine ligand to enhance lipolysis. Upon binding its ligand the β-
adrenergic receptor activates Gs (a heterotrimeric G protein embedded in the membrane) which in turn, via its alpha
subunit, stimulates adenylyl cyclise to produce c-AMP. This c-AMP then activates PKA which phosphorylates lipases
found in the adipose tissue to activate them for the breakdown of lipids. Each PKA is a holoenzyme and consists of a
regulatory subunit dimer with each regulatory subunit bound to a catalytic subunit. Under low levels of c-AMP the
holoenzyme remains intact and is catalytically inactive. When the level of c-AMP rises it binds to the two binding sites on
the regulatory subunits of PKA which causes them to release the catalytic subunits. For optimal function the catalytic
subunits must also be phosphorylated at the Thr 197 position allowing them to orientate catalytic residues properly at the
active site. The free catalytic subunits can then catalyse the transfer of ATP terminal phosphates to protein substrates at
serine or threonine residues. As well as increasing or decreasing the activity of a protein, PKA may also influence protein
synthesis. Firstly the PKA directly activates CREB (a transcription factor) which binds to certain sequences of DNA
called c-AMP response elements (CRE), altering the transcription of downstream genes and therefore protein synthesis.
PKA downregulation is achieved by a feedback mechanism. One of the substrates activated by PKA is a
phosphodiesterase which quickly converts c-AMP to AMP limiting the amount of c-AMP available for activation of PKA.
The catalytic subunit may also be downregulated itself by phosphorylation.

Immunological synapses
An immunological synapse is the interface between an antigen presenting cell (APC) and a lymphocyte i.e. T cell. The
APC, for example a macrophage or dendritic cell, displays peptides of foreign antigens via a major histocompatibility
complex (MHC) on its cell surface which is then recognised by the T-cell receptor (TCR). Unlike B cells, T cells are
unable to recognise antigens in the absence of antigen presentation. There are two types of MHC, class I and class II.
MHC class I displays intracellular antigenic peptides (mainly viruses) for activation of cytotoxic T-cells which directly
attack infected cells. MHC class II displays extracellular antigen peptides (bacteria, parasites etc) and activates helper and
regulatory T cells for amplification or suppression of the immune response, respectively. In the absence of an infection
MHC displays a self epitope which T cells do not normally respond to. Once activated the T cell then recognises the same
peptide:MHC complex on the surface of their target cells e.g. B cells, cytotoxic T cells or infected macrophages (in the
case of helper T cell activation) or any infected host cell (in the case of cytotoxic T cell activation). A large family of
MHC genes encode for MHC proteins, some of which are the most polymorphic known in higher vertebrates. As a result
it is very rare for two unrelated individuals to have an identical set of MHC proteins. This makes it very hard to find
matches for organ transplantations. MHC proteins are transmembrane heterodimers with extracellular N-terminal domains
which bind antigen for presentation. Each MHC is capable of binding a large number of different peptides. T cell
receptors are heterodimers. Most consist of an alpha and beta chain whereas a small percentage consists of a gamma and
delta chain. Each chain is composed of a variable and constant region. The constant region is responsible for anchoring
the receptor to the membrane and also includes a short cytoplasmic chain. The variable region is extracellular and is
responsible for binding the MHC:antigen complex. The variable domain at both the alpha and beta chain has three
complementarity determining regions (CDR) and an additional area of hyper-variability (HV4). HV4 does not generally
contact antigen. The residues are located at the interface of the alpha and beta chains and β-chain framework region which
is in close proximity to the CD3 signal transduction complex which propagates the signal into the cell. CDR3 is the main
CDR responsible for recognizing antigen although CDR1 of the α-chain and β-chain interact with the N-terminal and C-
terminal regions of the antigen peptide, respectively. CDR2 recognizes the MHC protein. The constant domains of the
TCR are linked by disulfide bonds. MHC proteins simultaneously bind co-receptors on the T cell to enhance the signal.
On helper T cells the co-receptor is CD4 and is specific for MHC class II. On cytotoxic T cells the co-receptor is CD8 and
is specific for MHC class I. The co-receptor not only ensures MHC specificity but also allows for prolonged engagement
between the APC and T cell allowing time for recruitment of essential signalling molecules e.g. protein kinases from
within the T cell. As well as the MHC:TCR interaction T cells require another signal to become fully activated. The
second signal, the co-stimulatory signal, is antigen non-specific and is provided by co-stimulatory molecules on the
surface of the APC and T cell. One of the best characterised co-stimulatory molecules expressed by T cells is CD28 which
interacts with CD80 and CD86 on the membrane of the APC. T cell co-stimulation is necessary for T cell proliferation,
differentiation and survival. Activation of T cells without co-stimulation may lead to T cell anergy, T cell deletion or the
development of immune tolerance.

Gluconeogenesis
Gluconeogenesis (GNG) is the metabolic process by which glucose, a major source of energy for the body, is generated
from non-carbohydrate carbon substances such as pyruvate, lactate and glycerol. It is one of two mechanisms used by
humans and many other animals to prevent hypoglycaemia (the lowering of blood glucose levels) and is a ubiquitous
process present in plants, animals, fungi, bacteria and other microorganisms. In vertebrates the process takes place mainly
in the liver but to a lesser extent also in the kidneys. The major inputs for gluconeogenesis come from the breakdown of
amino acids and from lactate produced by liver muscles and transported to the liver in the bloodstream. The first step in
GNG occurs in the mitochondria and involves the conversion of pyruvate (3C) to the intermediate oxaloacetate (4C). This
step requires energy (provided by ATP) and is catalysed by the enzyme pyruvate carboxylase. In order to get out of the
mitochondria oxaloacetate is converted to malate by malate dehydrogenase. Once there it is then converted back into
oxaloacetate by a cytoplasmic form of malate dehydrogenase. Next, phosphoenolpyruvate (PEP) carboxykinase catalyses
the conversion of oxaloacetate to phosphoenolpyruvate. This step also requires energy in the form of GTP. The process
proceeds by reversing the steps of glycolysis resulting in fructose-1,6-biphosphate which is then cleaved to produce
fructose-6-biphosphate. Fructose-6-biphosphate is then converted to glucose-6-phosphate by phosphoglucose isomerise
and all that remains is the simple cleavage of one phosphate to produce glucose. The entire process is summarised across.
The rate of gluconeogenesis is determined by the concentrations of lactate and other glucose precursors so that if the
process of glycolysis is highly active then gluconeogenesis in inactive and vice versa. Gluconeogenesis is also highly
controlled by AMP and citrate which inhibit and activate fructose-6-biphosphatase respectively. A high level of AMP
indicates that energy is low and there is a need for ATP generation. Conversely, high levels of ATP and citrate indicate
that energy is high and that biosynthetic intermediates are abundant. Under these conditions, glycolysis is nearly always
switched off and gluconeogenesis is promoted.

Transfer RNA
Transfer RNAs are approximately 75-90 nucleotides in size. Internal complementary basing causes the molecule to fold
into a structure in which open loops are connected to one another by double stranded regions. This gives the tRNA
molecule its characteristic clover leaf structure. The function of charged tRNA molecules in protein synthesis is to transfer
specific amino acids to the mRNA-ribosomal complex, so that a polypeptide chain can be assembled. The set of enzymes
responsible for charging tRNA are the Aminoacyl Synthetases.
One of the regions of tRNA used in the decoding operation is the Anticodon. This is essentially a sequence of three bases
that can base-pair with a codon sequence in the mRNA. Inside the ribosome, hydrogen binding of tRNA to mRNA holds
the amino acids in proximity to each-other, so that a peptide bond can be formed. No normal tRNA molecule has an
anticodon complementary to the codons UAG, UAA or UGA, thus these codons are stop signals.
Modifications to the base adjacent to the anticodon region in tRNA, has been shown to affect the speed and accuracy of
translation. However some codons cannot be translated if specific base modifications are not present on their cognate
tRNAs. For example, N37 modifications compensate for weak N36–N1 base pairs, by increasing stacking. Base
modifications help remove coding ambiguities and shape unstructured tRNA anticodon loops for translation.
Considering there are 4 types of bases, and each triplet encodes for an amino acid, you would expect there to be 61 types
of amino acid instead of 20 (because we already know UAG, UAA and UGA are stop codons). However this is not the
case. The wobble base theory set about solving this conundrum. The wobble base is the third base of a codon which is
less physically constrained than the first two. Take Serine for example, which is encoded by AAA and AAG and four
other triplet combinations. This shows that some amino acids are encoded for by more than one triplet. When an amino
acid has become attached to a tRNA, the tRNA is said to have become ‘charged’.
A second important site on a tRNA molecule is the Amino Acid Attachment Site. This is located at the 3’terminus of the
molecule, allowing its corresponding amino acid to base-pair to the mRNA codon covalently. A specific aminoacyl tRNA
synthetase matches each amino acid with its anticodon.

Helix loop helix

The helix-loop-helix motif was originally identified as the DNA-binding domain of phage-repressors. The HLH structure
comprises a stretch of 40-50 amino acids containing 2 amphipathic helices separated by a linker region (the loop) of
varying length. The larger of the two helices contains basic amino acid residues that interact with DNA, also known as the
‘Recognition helix’. βHLH proteins generally bind to a consensus DNA sequence called an E-box.
The recognition helix binds by a combination of hydrogen bonds and/or van der Waal interactions with the edges of
bases. The other α helix locks the recognition helix into position. MyoD is an example of a βHLH which is muscle-
specific.

TNF-alpha
Tumour necrosis factor-alpha (TNF-α) is a pleiotropic inflammatory cytokine. It possesses growth stimulating, regulatory
and inhibitory properties e.g. TNF-α induces neutrophil proliferation during inflammation, but it also induces neutrophil
apoptosis upon binding the TNF-R55 receptor. It is produced by several types of cells, particularly in macrophages. TNF-
α has an important role in the in the immune response against bacterial, fungal, viral, and parasitic invasions by acting as a
mediator of local inflammation. TNF-α is an acute phase protein that initiates a cascade of cytokine signalling, which
increases vascular permeability for the recruitment of macrophage and neutrophils to a site of infection. Macrophage
secreted TNF-α induces blood clotting which serves to contain infection. Without TNF-α, mice infected with gram
negative bacteria experience septic shock. TNF-α is also important for the necrosis of specific tumours.
The pathological activities of TNF-α have attracted much attention. Although TNF-α causes necrosis of some types of
tumors, it can promote the growth of some specific types of tumor cell. High levels of TNF-α have been shown to
correlate with an increased risk of mortality in cancer patients. TNF-α also participates in autoinflammatory and
autoimmune disorders such as RA. Anti-TNF drugs are currently in development to treat such disorders.

Telomeres
Where the genomes of Prokaryotes are circular, the chromosomes of humans and other eukaryotes are linear. The free
ends of linear DNA molecules introduce complications due to the fact that since polymerases act in a 5’-3’ manner
making complete replication of DNA ends difficult. Without telomerases, the lagging strand of the DNA would have an
incomplete 5’ end after the removal of the RNA primer. Each round of replication would thereby further shorten the
chromosome.
Telomeric DNA contains hundreds of tandem repeats of a 6 nucleotide sequence. One of the strands is G rich at the 3’ end
and it is slightly longer than the other strand. In us humans, the repeating G-rich sequence is AGGGTT. These repeated
sequences are formed by the enzyme telomerase which is essentially a specialised reverse transcriptase that carries its own
template. Telomerase is generally only expressed in high levels of rapidly growing cells. Thus it can play an important
role in cancer cell biology and in cell aging.
Telomeres protect the DNA ends by forming loop structures which are stabilised by many telomeric binding proteins.
These loops mask the ends of our chromosomes.

SH3 Domains
The SRC Homology 3 Domain (or SH3 domain) is a small protein domain of about 60 amino acids present in the non-
catalytic parts of enzymes such as phospholipase and several cytoplasmic tyrosine kinases such as Src. It has also been
identified in PI3 Kinase and Ras GTPase activating protein. Approximately 300 SH3 domains are found in proteins
encoded in the human genome.
The SH3 domain has a characteristic beta-barrel fold which consists of five or six β-strands arranged as two tightly packed
anti-parallel β sheets. The linker regions may contain short helices. The SH3-type fold is an ancient fold found in
eukaryotes as well as prokaryotes.
The classical SH3 domain is usually found in proteins that interact with other proteins and mediate assembly of specific
protein complexes, typically via binding to proline-rich peptides in their respective binding partner.

RNA
There are three types of RNA derived from hnRNA. mRNA contains the sequences of bases that specify the sequence of
amino acids. The amino acids specified by mRNA are attached to tRNA for entry into the ribosome and rRNA binds to a
set of proteins to form the ribosome machinery. Ribosomes provide binding sites for all accessory molecules necessary in
protein synthesis.
rRNA is generated from long precursor molecules called pre-rRNAs. The pre-rRNAs are cleaved by ribonucleases and
trimmed by exonucleases. rRNA synthesis occurs in the nucleolus with base and sugar modifications facilitated by small
nuclear RNAs (snRNA).
tRNA is an adaptor molecule that delivers amino acids to the ribosome and decodes the information in mRNA. They are
around 75-90 nucleotides long. Modified bases account for ~20% of the tRNA molecule. It has internal hydrogen bonding
to give it its characteristic clover structure.
Pre-mRNA must undergo various modifications before becoming mature mRNA. These include capping, tailing and
splicing.

Restriction endonucleases
RE’s are enzymes which cleave dsDNA in at a specific nucleotide pallindrome sequence. They are used in recombinant
DNA technology to insert a recombinant piece of DNA into a plasmid/vector. Transfection, transformation. EcoRI
GAATTC, BamH1 GGATCC. Sticky ends, blunt ends.

Phospholipase C
Phosphoinositol cascade…Angiotensin II activates GPCR. Gαq subunit activates PLC-β which cleaves PIP2 to form IP3
and DAG. IP3 increases Ca2+ and DAG activates PKC…mobilization of energy stores.
Classic activated RTKs recruit PLC-γ to their SH2 domains. PLC-gamma hydrolysed. Also cleaves PIP2 and DAG.

Peyer’s Patches
Peyer's patches are aggregations of lymphoid tissue that are usually found in the ileum. They differentiate the ileum from
the duodenum and jejunum. On average, about 30 are found in humans. Microscopically, Peyer’s patches appear as oval
or round lymphoid follicles. In adults, B lymphocytes are seen to predominate in the follicles' germinal centres whereas T
lymphocytes are found in the zones between follicles.
Because the lumen of the gastrointestinal tract is exposed to the external environment, much of it is populated with
potentially pathogenic microorganisms. Peyer's patches establish their importance in the immune surveillance of the
intestinal lumen and in facilitating the generation of the immune response within the mucosa. Pathogenic microorganisms
and other antigens entering the intestinal tract encounter macrophages, dendritic cells, B-lymphocytes, and T-lymphocytes
found in Peyer's patches. Peyer's patches are covered by a special epithelium that contains specialized cells called
microfold cells (M cells) which sample antigen directly from the lumen and deliver it to antigen-presenting cells (located
in a unique pocket-like structure on their basolateral side). B-cells and memory cells are stimulated upon encountering
antigen in Peyer's patches. These cells then pass to the mesenteric lymph nodes where the immune response is amplified.
Activated lymphocytes pass into the blood stream via the thoracic duct and travel to the gut where they carry out their
final effector functions.

PAMPS
Pathogen-associated molecular patterns, or PAMPs, are molecules associated with groups of pathogens that are
recognized by cells of the innate immune system. These molecules can be referred to as small molecular motifs conserved
within a class of microbes. They are recognized by Toll-like receptors (TLRs) and other pattern recognition receptors
(PRRs) in both plants and animals.
PAMPS activate innate immune responses, protecting the host from infection by identifying some conserved non-self
molecules. Bacterial Lipopolysaccharide (LPS), an endotoxin found on the bacterial cell membrane of a bacterium, is
considered to be the prototypical PAMP. LPS is specifically recognised by TLR 4, a recognition receptor of the innate
immune system. Other PAMPs include bacterial flagellin (TLR 5), peptidoglycan, and nucleic acid variants normally
associated with viruses, such as double-stranded RNA (dsRNA). Adjuvants in vaccines?

Oxaloacetate & the citrate cycle

Oxaloacetate (OAA) is a metabolic intermediate of the Citric Acid Cycle. In the first step of this cycle, OAA condenses
with Acetyl Co A to form Citrate with the use of the enzyme Citrate Synthase. Citrate goes on in the cycle to form many
intermediates through the use of different enzymes and redox reactions until Malate is formed. Malate can then undergo
oxidisation with NAD+ to reform OAA, hence completing the TCA cycle. (OAA+ Acetyl CoA-> Citryl CoA-> Citrate->
Isocitrate -> Oxalosuccinate -> alpha-Ketogluterate -> Succinyl CoA -> Succinate -> Fumerate ->Malate ->OAA.)
In some species, OAA is important in the Calvin cycle as it is converted to malate by an NADP-linked malate
dehydrogenase.
Aspartate and is also a precursor of OAA in transamination reactions. Aspartate + alpha-keto glutarate can form OAA and
glutamate which can then enter the TCA cycle as previously discussed. This reaction is reversible meaning that OAA is
indeed a precursor of aspartate.
Carboxylation of pyruvate to oxaloacetate (OAA) by pyruvate dehydrogenase kinase is one of the 3 alternate fates of
pyruvate at the end of glycolysis. This reaction is important because it replenishes the citric acid cycle intermediates and
provides substrates for gluconeogenesis.
In the first step of gluconeogenesis, pyruvate carboxylase converts pyruvate to OAA which is a precursor of
phosphoenolpyruvate through the use of the enzyme PEPCK.

NFkB
NFkB is a very important molecule of the immune system. It binds to the enhancer gene sequence of the kappa light chain
in B cells. NFkB is activated by stress/danger to a cell e.g. immunecomprimisation or UV rays. It forms dimers with two
other proteins subunits p50 and p65. NFkB is pre-formed in the cell and hence is very fast acting. It is kept inactive in the
cell by the IkB inhibitory subunit. The inhibitory IkB subunit can be degraded by IL-2, allowing for the p65 region to get
phosphorylated. Once phosphorylated, the p65 subunit allows the complex to translocate to the nucleus to initiate
transcription of IL-8?
NFkB is also an anti-apoptotic factor. The kinase which activates NFkB is known as IKK. IKK has 3 subunits: α, β and γ.
IKK is an important drug target against inflammation. IKK is regulated by many kinases, which is why some alternative
pathways such as the IkB-alpha, P50 P65 can also get activated. The IFN-β promoter of viruses is NFkB dependant. The
NFkB promoter sequence is the same as the sequence of the HIV promoter. HIV can thereby damage cells by recruiting
the NFkB TF for transcription of their own genes.
Nucleophiles and Electrophiles
A nucleophile is a species that donates an electron-pair to an electrophile, (see later) forming a chemical bond in a
reaction. All molecules or ions with a free pair of electrons or at least one pi bond can act as nucleophiles. Because
nucleophiles donate electrons, they are by definition Lewis bases.
Nucleophiles are electron rich. Nucleophilic character describes the affinity of a nucleophile to the nuclei. Nucleophiles
may take part in nucleophilic substitution, whereby a nucleophile becomes attracted to a full or partial positive charge. In
the example below, the OH- is the nucleophile.

Electrophiles are usually positively charged species that are attracted to an electron rich centre, (can also be uncharged
species such as a Lewis acid). In chemistry, an electrophile participates in a chemical reaction by accepting an electron
pair in order to bond to a nucleophile. Because electrophiles accept electrons, they are Lewis acids. Most electrophiles are
completely positively charged, have an atom that carries a partial positive charge or have an atom that does not have an
octet of electrons.
Electrophiles attack the most electron-populated part of one nucleophile. (Markovnikov's rule) The electrophiles
frequently seen in the organic syntheses are cations such as H +, polarized neutral molecules such as HCl, alkyl halides,
and carbonyl compounds and polarizable neutral molecules such as Cl2 and Br2.

Neutrophils
Neutrophils are produced in the bone marrow and circulate in the blood. They are the most common types of white blood
cell comprising about 50-70% of all WBCs. They form part of the polymorphonuclear cell family (PMNs) together with
basophils and eosinophils. Neutrophils are phagocytic, meaning that they can ingest antigenic molecules, although they do
not survive the act. The internalization of foreign bodies results in the formation of a phagosome into which reactive
oxygen species and hydrolytic enzymes are secreted (e.g. NADPH oxidase). The consumption of oxygen during the
generation of reactive oxygen species has been termed the "respiratory burst”. Neutrophils are the first immune cells to
arrive at the site of infection through chemotaxis gradients of molecules such as IL-8 or IFN-γ. Neutrophils are short lived
with a half-life of 4-10 hours when not activated. Neutrophils are part of the innate immune system, which means that
they can non-specifically destroy any invaders that they encounter in the body, such as bacteria and parasites. They are the
predominant cells in pus, accounting for its whitish/yellowish appearance.

mtDNA
Mitochondrial DNA (mtDNA) is the DNA located in organelles called mitochondria. Mitochondria are structures within
eukaryotic cells that convert the chemical energy from food into a form that cells can use, adenosine triphosphate (ATP).
Most other DNA present in eukaryotic organisms is found in the cell nucleus. Mitochondrial DNA can be regarded as the
smallest chromosome, and was the first significant part of the human genome to be sequenced. In most species, including
humans, mtDNA is inherited solely from the mother.
The DNA sequence of mtDNA has been determined from a large number of organisms and individuals (including some
organisms that are extinct), and the comparison of those DNA sequences allows biologists to elucidate the evolutionary
relationships among species. It also permits an examination of the relatedness of populations, and so has become
important in anthropology and field biology. Nuclear and mitochondrial DNA is thought to be of separate evolutionary
origin, with mtDNA being derived from the circular genomes of the bacteria that were engulfed by the early ancestors of
today's eukaryotic cells. This theory is called the endosymbiotic theory.

Mast Cells
Mast cells are found resident in tissues throughout the body, particularly in association with blood vessels and nerves, and
in proximity to surfaces that interface the external environment. Mast cells are bone marrow-derived and particularly
depend upon stem cell factor for their survival. Mast cells express a variety of phenotypic features within tissues as
determined by the local environment. Withdrawal of required growth factors results in mast cell apoptosis.
Mast cells may be activated by a number of receptors leading to distinct signalling pathways. After activation, mast cells
may immediately extrude granule-associated mediators and generate lipid-derived substances that induce immediate
allergic inflammation such as histamine. Mast cell activation can also be triggered by the synthesis of chemokines and
cytokines. Biological functions of mast cells appear to include a role in innate immunity, involvement in host defence
mechanisms against parasitic infestations, immunomodulation of the immune system, and tissue repair and angiogenesis. \
mast cells hve been implicated in the pathogenesis of diseases such as fibrosis.

IFN-γ
Interferon-gamma is a dimerized soluble cytokine that is the only member of the type II class of interferons. In humans,
the IFN-γ protein is encoded by the IFNG gene. IFN-γ is a cytokine that is critical for innate and adaptive immunity
against viral and intracellular bacterial infections as well as tumour control. Aberrant IFN-γ expression is associated with
a number of autoimmune diseases. The importance of IFN-γ in the immune system stems in part from its ability to inhibit
viral replication directly and most importantly from its immune-stimulatory and immune-modulatory effects. IFN-γ is
produced predominantly by natural killer (NK) and natural killer T cells (NKT) as part of the innate immune response,
and by CD4 Th1 and CD8 cytotoxic T lymphocyte (CTL) effector T cells once antigen-specific immunity develops. The
IFN-γ monomer consists of a core of six α-helices. IFN-γ signals through the JAK-STAT pathway.

Hydrogen bonds
A hydrogen bond is the attractive interaction of a hydrogen atom with an electronegative atom, such as nitrogen, oxygen
or fluorine. The hydrogen must be covalently bonded to another electronegative atom in order for the bond to form. These
bonds can occur between molecules (intermolecularly), or within different parts of a single molecule (intramolecularly).
The hydrogen bond (5-30 kJ/mole) is stronger than a van der Waals interaction, but weaker than covalent or ionic bonds.
This type of bond occurs in both inorganic molecules such as water and organic molecules like DNA.
A hydrogen atom attached to a relatively electronegative atom is a hydrogen bond donor, e.g. fluorine, oxygen or
nitrogen. An example of a hydrogen bond donor is ethanol, which has hydrogen bonded to oxygen. An electronegative
atom such as fluorine, oxygen, or nitrogen is always considered a hydrogen bond acceptor, regardless of whether it is
bonded to a hydrogen atom or not. The hydrogen bond is often described as an electrostatic dipole-dipole interaction. The
most ubiquitous, and perhaps simplest, example of a hydrogen bond is found between water molecules. In a discrete water
molecule, there are two hydrogen atoms and one oxygen atom.
Hydrogen bonding plays an important role in determining the 3D structures adopted by proteins and nucleic bases. In
these macromolecules, bonding between parts of the same macromolecule cause folding which helps determine the
molecule's physiological or biochemical role. The double helical structure of DNA for example, is due largely to
hydrogen bonding between the base pairs which link one complementary strand to the other and enable replication.

Factors increasing membrane fluidity

The presence of Cholesterol (increases and decreases), Temperature (increasing temp increases fluidity), composition
of Phospholipids (degree of saturation...unsaturated fatty acids increase membrane fluidity and lower its phase transition
temperature due to double bonds causing bending of fatty acid molecules) and type of membrane proteins (integral,
transmembrane and peripherial).
Glycogen
Glycogen is a molecule that serves as the secondary long-term energy storage in most eukaryotic cells, with the primary
energy stores being held in adipose tissue. Glycogen is made primarily by the liver hepatocytes and muscle cells, but it
can also be made by glycogenesis within the brain and stomach. The uterus stores glycogen during pregnancy to nourish
the embryo. Glycogen is an analogue of starch. It is found in granules in the cell cytosol/cytoplasm in many cell types,
and it plays an important role in the glucose cycle. Glycogen forms an energy reserve that can be quickly mobilized to
meet a sudden need for glucose, but one that is less compact than the energy reserves of triglycerides. IL-6, adrenalin,
cortisol and growth factor all help to mobilise glycogen stores.
Glycogenolysis helps to keep blood sugar levels regulated. Glycogen is cleaved by the enzyme glycogen phosphorylase to
produce monomers of glucose-1-phosphate. These monomers are then converted to glucose 6-phosphate by
phosphoglucomutase in glycolysis. The most common disease in which glycogen metabolism becomes abnormal is
diabetes, in which, because of abnormal amounts of insulin, liver glycogen can be abnormally accumulated or depleted.

Insulin is a hormone that regulates the concentration of glucose in the blood. A high fasting blood sugar level is an
indication of pre-diabetic/diabetic conditions.
The first step of glycoclysis is the phosphorylation of glucose by the enzyme hexokinase to prepare it for later breakdown
provide energy (net 2 molecules of ATP).

Importance of G-C bonds

DNA consists of multiple nucleotides linked together in a double helix strand. Nucleotides consist of sugar (ribose) bases
(deoxyribose) and a phosphate. Nucleosides are the same as Nucleotides but without the phosphate. There are four types
of nucleotide base that can found in DNA, all in equal ratios to one another; Adenine, Thymidine, Guanine and Cysteine.
A and G are Purines and G and C are Pyridimines.
The GC pair is bound by three hydrogen bonds, while AT pairs are bound by two hydrogen bonds. DNA with high GC-
content is more stable than DNA with low GC-content. In PCR experiments, the GC-content of primers are used to predict
their annealing temperature to the template DNA. A higher GC-content level indicates a higher melting temperature.
Purines always bond to Pyridimines by complementary base pairing to ensure that the molecule is guaranteed to be the
same size all over and that the amount of one base is equal to the amount of its complementary base. This condition is
known as Chargaff's rules. The GC box, which as the name suggests consists solely of G-C bases, is an important
transcription factor. It is primarily located 100-150bp upstream from the TATA box.

Epitopes
An epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system,
specifically by antibodies, B cells, or T cells. The part of an antibody that recognizes the epitope is called a paratope.
Although epitopes are usually thought to be derived from non-self-proteins, some sequences derived from the host that
can be recognized are also classified as epitopes.
T cell epitopes are presented on the surface of an antigen-presenting cell, where they are bound to MHC molecules. T cell
epitopes presented by MHC class I molecules are typically peptides between 8 and 11 amino acids in length. MHC class II
molecules present longer peptides, 13-17 amino acids in length and non-classical MHC molecules also present non-
peptidic epitopes such as glycolipids.
Epitopes can be mapped using protein microarrays, and with ELISA. Intensive research is currently taking place to design
reliable tools that will predict epitopes on proteins. Epitope tags are often used in proteomics and the study of other gene
products. Using recombinant DNA techniques genetic sequences coding for epitopes that are recognized by common
antibodies can be fused to the gene. Following synthesis, the resulting epitope tag allows the antibody to find the protein
or other gene product enabling lab techniques for localization, purification, and characterization. Common epitopes used
for this purpose are Myc-tag, GST-tag and His tags.
Entropy
Entropy is a thermodynamic property that can be used to measure the disorder of a system. In classical thermodynamics,
the concept of entropy is defined by the second law of thermodynamics, which states that the entropy of an isolated
system always increases or remains constant. An air conditioner, for example, may cool the air in a room, thus reducing
the entropy of the air of that system. The heat expelled from the room (the system), which the air conditioner discharges to
the outside air, will always make a bigger contribution to the entropy of the environment than will the decrease of the
entropy of the air of that system. Thus, the total of entropy of the room plus the entropy of the environment increases, in
agreement with the second law of thermodynamics.

DAG
The phosphoinositiol cascade is a GPCR controlled pathway which is activated by hormones such as vasopressin or
angiotensin II. In this pathway, the enzyme PLC-γ cleaves PIP2 into two important secondary messengers, IP3 and DAG.
IP3 is a diffusible secondary messenger that functions to increase the cellular levels of Ca 2+ whereas DAG is a stationary
messenger that remains in the inner layer of the plasma membrane. DAG functions to recruit Protein Kinase C (PKC) -a
calcium-dependent kinase that phosphorylates many other proteins that bring about changes in the cell.

Beer-Lambert Law
The Beer-Lambert law is the linear relationship between absorbance and concentration of an absorbing species. The
general Beer-Lambert law is usually written as:
A = ecl
Where A is the measured absorbance, e absorptivity coefficient, l is the path length, and c is the concentration of the
solution. The wavelength-dependent molar absorptivity coefficient units are M-1 cm-1.

Acetyl Co-A
Acetyl-CoA is an important molecule in metabolism. Its main function is to convey carbon atoms within the acetyl group
to the citric acid cycle for oxidization and energy production. Acetyl-CoA is the thioester between coenzyme A (a thiol)
and acetic acid (an acyl group carrier). Acetyl-CoA is produced during the second step of aerobic cellular respiration,
pyruvate decarboxylation, which occurs in the matrix of the mitochondria. Acetyl-CoA then enters the citric acid cycle.
The oxidative conversion of pyruvate into acetyl-CoA is referred to as the pyruvate dehydrogenase reaction. Acetyl CoA
has an important role in ketone body synthesis.

2017
SH2 and SH3 domains
● Identified as a region of homology between two oncogenic tyrosine kinases that lay outside the catalytic domain. They termed
this the Src homology 2, or SH2, domain.
● There are over 100 proteins that contain SH2 and SH3 domains.
● SH2 and SH3 domains are small protein modules that mediate protein-protein interactions in signal transduction pathways that
are activated by protein tyrosine kinases.
● SH2 domains bind to short phosphotyrosine-containing sequences in growth factor receptors and other phosphoproteins.
● SH3 domains bind to target proteins through sequences containing proline and hydrophobic amino acids.
● SH2 and SH3 domain containing proteins, such as Grb2 and phospholipase C gamma, utilize these modules in order to link
receptor and cytoplasmic protein tyrosine kinases to the Ras signaling pathway and to phosphatidylinositol hydrolysis,
respectively.
IRF3
● Interferon regulatory factor is a member of the interferon regulatory transcription factor (IRF) family.
● IRF3 plays an important role in the innate immune system's response to viral infection. It is involved in Toll-like receptor
signalling pathways.IRF3 is localized in the cytoplasm in a latent form.
● Upon phosphorylation by TBK1 or IKKε, IRF3 forms a homodimer, followed by translocation into the nucleus. An IFN
enhanceosome is then assembled consisting of IRF3, NF-κB and activating transcription factor 2 (ATF2)–C-JUN heterodimers
that recruit the histone acetyltransferase CREB-binding protein (CBP) to the IFNB promoter. IRF3 also binds to the IFNa
promoter.
● IRFs and IFNs have been implicated in eliciting anti-tumor effects.
● HCMV UL82 also prevents STING from interacting with TBK1 and IRF3, which are necessary for cGAMP signaling.
Eosinophils
● Eosinophils are granulocytes that were first described to stain with acid aniline dyes, such as eosin.
● Blood and tissue eosinophilia are hallmark signs of helminth infection, allergy, asthma, eosinophilic gastrointestinal disorders,
and a number of other rare disorders.
● Human eosinophils have a bilobed nucleus with highly condensed chromatin and 2 major types of granules, specific and
primary.
● A major cationic protein in the specific granules are major basic protein (MBP) to activate neutrophils.
● Eosinophils express an array of cell-surface molecules, including immunoglobulin receptors for IgG (FcgRII/CD32) and IgA
(FcaRI/CD89); complement receptors (CR1/CD35, CR3, and CD88); cytokine receptors (IL-3R, IL-5R, and GM-CSF that
promote eosinophil development, as well as receptors for IL-1a, IL-2, IL-4, IFN-a, and TNF-a); chemokines (CCR1 and CCR3);
adhesion molecules (very late antigen 4, a4b7); and TLRs (particularly TLR7/8).
● Eosinophils are activated through receptors to release their cytotoxic granules through piecemeal degranulation.
● Eosinophils develop in the bone marrow and are released into the circulation, most notably after stimulation by IL-5 priming
them to enter the GI or mucosa.
● Eosinophils can express a range of cytokines, their production of cytotoxic granule proteins is thought to be their major effector
function. They secrete IL-4 and IL-13 to activate M1 macrophages and also prime B cells to secrete IgG and recruit Th2 cells.
MDSCs
● Myeloid-derived suppressor cells are are immunosuppressive cells from the myeloid lineage.
● They are common in pathological conditions such as chronic infections and cancer.
● They are promoted by inhibitory cytokines such as IL-10 and IL-13 and growth factors GM-CSF and VEGF. They are
suppressors of T cells, particularly CD8+ cytotoxic T cells.
● MDSCs accumulate in the lymph nodes (LNs), spleen, and liver of tumor-bearing mice and humans where they contribute to
tumor evasion of cell-mediated immunity.
● Among the mechanisms proposed for the immune-suppressive properties of MDSCs, L-arginine catabolism appears to be
important in MDSC-induced T-cell dysfunction. MDSCs can express both arginase-1 and inducible nitric oxide synthase
(iNOS), both of which metabolize L-arginine, leading to the production of the byproducts urea, L-ornithine, and citrulline and
nitric oxide, respectively. L-arginine deprivation results in repressed expression of the T cell–signaling molecule, CD3ζ, as well
as T-cell cycle arrest.
● They have recently been studied in their inhibition of GVHD.

Glycosylation
● Glycosylation is the attachment of sugar moieties to proteins, is a post-translational modification (PTM) that provides greater
proteomic diversity than other PTMs.
● Glycosylation is critical for a wide range of biological processes, including cell attachment to the extracellular matrix and
protein–ligand interactions in the cell.
● This PTM is characterized by various glycosidic linkages, including N-, O- and C-linked glycosylation, glypiation (GPI anchor
attachment), and phosphoglycosylation.
● Over 40 disorders of glycosylation have been reported in humans.
● Glycosylation is often used by viruses to shield the underlying viral protein from immune recognition. A significant example is
the dense glycan shield of the envelope spike of the human immunodeficiency virus.

Muckle-Wells Syndrome
● Muckle-Wells syndrome (MWS) is a autosomal dominant disorder characterized by periodic episodes of skin rash, fever, and
joint pain.
● Progressive hearing loss and kidney damage also occur in this disorder.
● Mutations in the NLRP3 gene (also known as CIAS1) cause Muckle-Wells syndrome.
● The NLRP3 gene provides instructions for making a protein called cryopyrin.
● Cryopyrin belongs to a family of proteins called nucleotide-binding domain and leucine-rich repeat containing (NLR) proteins.
● These proteins are involved in the immune system, helping to regulate the process of inflammation.
● Cryopyrin is involved in the assembly of a molecular complex called an inflammasome, which helps trigger the inflammatory
process. Researchers believe that NLRP3 gene mutations that cause Muckle-Wells syndrome result in a hyperactive cryopyrin
protein and an inappropriate inflammatory response.
● Impairment of the body's mechanisms for controlling inflammation results in the episodes of fever and damage to the body's
cells and tissues seen in Muckle-Wells syndrome.

NADH
● Nicotinamide adenine dinucleotide (NAD) is a coenzyme found in all living cells.
● Nicotinamide adenine dinucleotide exists in two forms: an oxidized and reduced form abbreviated as NAD+ and NADH
respectively.
● In metabolism, the compound accepts or donates electrons in redox reactions.
● Energy is transferred to NAD+ by reduction to NADH, as part of beta oxidation, glycolysis, and the citric acid cycle.
● In eukaryotes the electrons carried by the NADH that is produced in the cytoplasm are transferred into the mitochondrion (to
reduce mitochondrial NAD+) by mitochondrial shuttles, such as the malate-aspartate shuttle.
● The mitochondrial NADH is then oxidized in turn by the electron transport chain, which pumps protons across a membrane
and generates ATP through oxidative phosphorylation.
Dendritic Cell
● Professional antigen presenting phagocytes of the innate immune system.
● Named after their resemblance of dendrites on nerve cells in the brain.
● Act as a bridge between the innate and adaptive immune system, through antigen presentation to T cells in the lymph nodes.
● Have a variety of PRR’s (TLRs, Nod-like, RIG-I, CLRs) that detect PAMPs and present these antigen peptides on MHC I or II
to CD8+ or CD4+ T cells in the lymph nodes respectively.
● Once activated they lose their phagocytic ability and increase co-stimulatory receptor expression (e.g. CD80/CD86)
● Derive from a myeloid progenitor cell, and share a common myeloblast and monocyte progenitor cell with macrophages.
● Monocytes that can be split into two groups conventional and plasmacytoid.
● Tissue resident types include Langerhaans cells in the skin and mucosal CD103 DCs.
● Have been investigated in cancer vaccine use, injecting DC’s with loaded tumour peptide.

Actin
● is a family of globular multi-functional proteins that form microfilaments.
● It can be present as either a free monomer called G-actin (globular) or as part of a linear polymer microfilament called F-actin
(filamentous)
● Essential for such important cellular functions as the mobility and contraction of cells during cell division.
● Participates in many important cellular processes, including muscle contraction, cell motility, cell division and cytokinesis,
vesicle and organelle movement, cell signaling, and the establishment and maintenance of cell junctions and cell shape.
● ATP is required in order to maintain its structural integrity.
● Actin is one of the most abundant proteins in eukaryotes, where it is found throughout the cytoplasm.
● The Arp2/3 complex binds to actin filaments at 70 degrees to form new actin branches off existing actin filaments. Arp2/3-
mediated nucleation is necessary for directed cell migration.
● The growth of actin filaments can be regulated by thymosin and profilin.
● Myofibrils are made of thin filaments of actin, and thick filaments of the motor-protein myosin. These myofibrils use energy
derived from ATP to create movements of cells, such as muscle contraction.

Thymic Selection
● Differentiated into positive selection and negative selection
● A mechanism of central tolerance
● Also known as T cell education
● Occurs after VDJ recombination and TcR creation
● Positive selection ensures that aB T cells can recognise MHC I or II, those that do not recognise self-peptide on MHC are
eliminated
● Negative selection then occurs, to eliminate any autoreactive single positive T cells, or T cells that bind MHC with self-peptide
too strongly
● These autoreactive T cells undergo apoptosis, those that don’t lead to the risk of autoimmune diseases
● Only 3% of T cells from the thymic selection process leave the thymus as naive mature T cells and enter the periphery and
lymph nodes to await antigen presentation

NLRP3
● Nod-like receptor protein 3
● Made up of three domains: a C terminal leucine rich repeat (LRR), NACHT (common to all NOD-like receptors) and PYD (pyrin
domain)
● Mutations in NLRP3 gene give rise to auto-inflammatory diseases CAPS (cryopyrin-associated periodic syndrome
● Activated by multiple mechanisms, by PAMPs and DAMPs, by cathepsin B from a ruptured lysosome, ROS, K+ efflux etc.
● Forms the inflammasome cleaving pro-caspase 1 to caspase 1 and IL-1B, IL-18 production.

Calmodulin
● CAM, calcium modulated protein
● Calcium binding modulator protein found in all eukaryotic cells
● Binding of Ca2+ is necessary for calmodulin activation
● Once bound, it acts as part of a calcium signal transduction pathway
● Involved in apoptosis, inflammation, metabolism, smooth muscle contraction
● In TcR signalling, Ca2+ activated calmodulin works with DAG to activate PKC and RasGRP. PKC activates CARMA which
leads to IKK recruitment and NFkB activation.
IL-10
● Interleukin 10, also known as human cytokine synthesis inhibitory factor (CSIF)
● Important anti-inflammatory cytokine
● Signals through JAK1, TYK2 and STAT3
● Has multiple, pleiotropic, effects in immunoregulation and inflammation.
● Downregulates the expression of Th1 cytokines, MHC class II antigens, and co-stimulatory molecules on macrophages.
● It also enhances B cell survival, proliferation, and antibody production.
● IL-10 can block NF-κB activity, and is involved in the regulation of the JAK-STAT signaling pathway.
Angiogenesis
● is the physiological process through which new blood vessels form from pre-existing vessels.
● Angiogenesis is a normal and vital process in growth and development, as well as in wound healing and in the formation of
granulation tissue.
● It is also a fundamental step in the transition of tumors from a benign state to a malignant one, leading to the use of
angiogenesis inhibitors in the treatment of cancer.
● Stimulator molecules of angiogenesis during tumour progression include VEGF, TGF-B, integrins and histamine.
● Vascular endothelial growth factor (VEGF) has been demonstrated to be a major contributor to angiogenesis, increasing the
number of capillaries in a given network.

2016
Tryptophan
It contains an α-amino group, an α-carboxylic acid group, and a side chain indole, making it a non-polar aromatic amino acid. It is
essential in humans, meaning the body cannot synthesize it: it must be obtained from the diet. Tryptophan is also a precursor to the
neurotransmitter serotonin and the hormone melatonin.

IL-23
● Interleukin-23 (IL-23) is a heterodimeric cytokine composed of an IL12B (IL-12p40) subunit (that is shared with IL12) and the
IL23A (IL-23p19) subunit.
● Prior to the discovery of IL-23, IL-12 had been proposed to represent a key mediator of inflammation in mouse models of
inflammation.
● The discovery of an additional potential binding partner for IL-12p40 led to a reassessment of this role for IL-12.
● Seminal studies in experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis, showed that IL-23 was
responsible for the inflammation observed, not IL-12 as previously thought.
● p19 subunit targeted in MS therapy, Tildrakizumab and Guselkumab: block yd T cells hence Th17s, by not blocking CD4+ T
cells, we are not compromising the immune system functions, less side effects.

Metformin
● Metformin, marketed under the trade name Glucophage among others, is the first-line medication for the treatment of type 2
diabetes, particularly in people who are overweight.
● Inhibits oxygen consumption in tumor cells in vitro and in vivo, resulting in reduced intratumoral hypoxia.
●

PAMPs
● Pathogen associated molecular patterns
● Non-self molecules recognised by the immune system to protect the host from danger
● Bind pathogen recognition receptors e.g. TLRs on innate cells to activate them such as macrophages and DCs
● These molecules can be referred to as small molecular motifs conserved within a class of microbes.
● Bacterial lipopolysaccharides (LPS), endotoxins found on the cell membranes of gram-negative bacteria,are considered to be
the prototypical class of PAMPs.
● LPS is recognised by TLR4
● Other PAMPs include bacterial flagellin (recognized by TLR5), lipoteichoic acid from gram-positive bacteria (TLR2/6) and
nucleic acid variants normally associated with viruses, such as double-stranded RNA (dsRNA) recognized by TLR3 or
unmethylated CpG motifs, recognized by TLR9.
● Bind TLRs and activate signalling cascades such as JAK STAT to induce effector responses

Neutrophils
● Important granulocytes of the innate immune system
● They form part of the polymorphonuclear cells family (PMNs) together with basophils and eosinophils.
● Neutrophils are the most abundant type of granulocytes and the most abundant (40% to 70%) type of white blood cells in most
mammals.
● The nucleus has a characteristic lobed appearance, the separate lobes connected by chromatin.
● They are formed from stem cells in the bone marrow.
● They are short-lived and highly motile.
● Neutrophils are named as such because they stain a neutral pink colour in Wright’s-Giemsa staining.
● Neutrophils are one of the first-responders of inflammatory cells to migrate towards the site of inflammation. They migrate
through the blood vessels, then through interstitial tissue, following chemical signals such as Interleukin-8 (IL-8), C5a and
Leukotriene B4 in a process called chemotaxis.
● They are the predominant cells in pus, accounting for its whitish/yellowish appearance.
 ● They extravate from the blood vessels through rolling adhesion model and enter the tissues by diapedesis using adhesion
receptors E-cadherin that binds Sialyl-Lewis X, and LFA-1 and I-CAM.
● Recruited by Th17 cells through the secretion of IL-17F and GM-CSF and are responsible for extensive tissue damage during
autoimmune disorders.
● They can internalize and kill many microbes, each phagocytic event resulting in the formation of a phagosome into which
reactive oxygen species and hydrolytic enzymes are secreted in a process known as respiratory burst.
● They can release an assortment of cytotoxic granules including cathelicidins, cathepsin, collagenase, lactoferrin, histamine
and defensins.
● Trojan Horse S.aureus immune evasion strategy

Warburg Effect
● The Warburg effect is the enhanced conversion of glucose to lactate observed in tumor cells, even in the presence of normal
levels of oxygen.
● Otto Heinrich Warburg demonstrated in 1924 that cancer cells show an increased dependence on glycolysis to meet their
energy needs, regardless of whether they were well-oxygenated or not.
● Converting glucose to lactate, rather than metabolizing it through oxidative phosphorylation in the mitochondria, is far less
efficient as less ATP is generated per unit of glucose metabolized.
● Therefore, a high rate of glucose uptake is required to meet increased energy needs to support rapid tumor progression.
● Aerobic glycolysis supports various biosynthetic pathways and, consequently, the metabolic requirements for proliferation.
● The PI3K pathway is considered to be a major determinant of the glycolytic phenotype through AKT1 and mTOR signaling,
and subsequent downstream Hypoxy-Inducible Factor 1 (HIF-1) transcription factor activation.

Phosphatidylserine
● is a phospholipid and is a component of the cell membrane.
● It plays a key role in cell cycle signaling, specifically in relationship to apoptosis.
● It consists of two fatty acids attached in ester linkage to the first and second carbon of glycerol and serine attached through a
phosphodiester linkage to the third carbon of the glycerol.
● When the phosphatidylserines flip to the extracellular (outer) surface of the cell, they act as a signal for macrophages to engulf
the cells.
● Phosphatidylserine plays a role in blood coagulation (also known as clotting). When circulating platelets encounter the site of
an injury, collagen and thrombin -mediated activation causes externalization of phosphatidylserine (PS) from the inner
membrane layer, where it serves as a pro-coagulant surface.

Hydrophobic effect
● The hydrophobic effect is the observed tendency of nonpolar substances to aggregate in an aqueous solution and exclude
water molecules.
● The hydrophobic effect is responsible for the separation of a mixture of oil and water into its two components. It is also
responsible for effects related to biology, including: cell membranes and vesicles formation, protein folding, insertion of
membrane proteins into the nonpolar lipid environment and protein-small molecule associations.
ILCs
● are a group of innate immune cells that are derived from common lymphoid progenitor (CLP) and belong to the lymphoid
lineage.
● This relatively newly described group of cells has varying physiological functions; some functions are analogous to helper T
cells, while the group also includes cytotoxic NK cells.
● Accordingly, they have an important role in protective immunity and the regulation of homeostasis and inflammation, so their
dysregulation can lead to immune pathology such as allergy, bronchial asthma and autoimmune disease.
● Cancer immunosurveillance
● Group 1 ILCs constitutively express transcription factor T-bet and is able to produce Th1 cytokines (notably IFNγ and TNF)
after stimulation with IL-12 or IL-18. ILC1 cells comprise NK cells.
● ILC2s (also termed natural helper cells, nuocytes, or innate helper 2 cells) play the crucial role of secreting type 2 cytokines in
response to helminth infection. They have also been implicated in the development of allergic lung inflammation.
● They require IL-7 for their development, which activates two transcription factors (both required by these cells)—RORα and
GATA3. After stimulation with Th2 polarising cytokines (e.g. IL-25, IL-33, TSLP) ILC2s start to produce IL-5, IL-13, IL-9, IL-4.
ILC2s are critical for primary responses to local Th2 antigens e.g. helminths and viruses and that is why ILC2s are abundant in
tissues of skin,lungs, livers and gut.
● Group 3 ILCs are defined by their capacity to produce cytokines IL-17A and/or IL-22. They are the innate counterpart to T h17
cells, and share the common transcription factor of RORγt. They comprise ILC3s and lymphoid tissue-inducer (LTi) cells.

microRNA
● Also called miRNA
● a small non-coding RNA molecule (containing about 22 nucleotides) found in plants, animals and some viruses, that functions
in RNA silencing and post-transcriptional regulation of gene expression.
● Encoded by eukaryotic nuclear DNA in plants and animals and by viral DNA in certain viruses whose genome is based on
DNA, miRNAs function via base-pairing with complementary sequences within mRNA molecules.
● As a result, these mRNA molecules are silenced, by one or more of the following processes: Cleavage of the mRNA strand
into two pieces,destabilization of the mRNA through shortening of its poly(A) tail, and, less efficient translation of the mRNA
into proteins by ribosomes.
Autophagosome
● An autophagosome is a spherical structure with double layer membranes. It is the key structure in macroautophagy, the
intracellular degradation system for cytoplasmic contents (e.g., abnormal intracellular proteins, excess or damaged organelles)
and also for invading microorganisms.
● After formation, autophagosomes deliver cytoplasmic components to the lysosomes. The outer membrane of an
autophagosome fuses with a lysosome to form an autolysosome.
● The lysosome's hydrolases degrade the autophagosome-delivered contents and its inner membrane.
● The initial step of autophagosome formation of an omegasome on the endoplasmic reticulum, followed by of elongation of
structures called phagophores.
● The formation of autophagosomes is controlled by Atg genes.
● Autophagy allows the orderly degradation and recycling of cellular components.
● In disease, autophagy has been seen as an adaptive response to stress, which promotes survival, whereas in other cases it
appears to promote cell death and morbidity. In the extreme case of starvation, the breakdown of cellular components
promotes cellular survival by maintaining cellular energy levels.
● Important in TB immune response, evaded by the mycobacterium
● 2016 Nobel Prize in Physiology or Medicine to Japanese autophagy researcher Yoshinori Ohsumi.

Gamma delta T cells

● Gamma delta T cells (γδ T cells) are T cells that have a distinctive T-cell receptor (TCR) on their surface. Most T cells are αβ
(alpha beta) T cells with TCR composed of two glycoprotein chains called α (alpha) and β (beta) TCR chains. In contrast,
gamma delta (γδ) T cells have a TCR that is made up of one γ (gamma) chain and one δ (delta) chain.
● This group of T cells is usually much less common than αβ T cells, but are at their highest abundance in the gut mucosa,
within a population of lymphocytes known as intraepithelial lymphocytes (IELs).
● RORyt, Tbet, Foxp3 plasticity allows them to vary receptor expression.
● yD T cells activation is not restricted by MHC recognition therefore can provide rapid effector responses against infection.
● They are activated by the secretion of IL-1B from monocytes, and secrete a similar repertoire of cytokines to Th17 cells: IL-
17A, IL-17F, IL-21, IL-22, IFNy, GM-CSF, TNFa
● They are implicated in autoimmune diseases such as psoriasis and MS as they are potent activators of Th17 cells.

DNA polymerase
● DNA polymerases are enzymes that synthesize DNA molecules from deoxyribonucleotides, the building blocks of DNA.
● These enzymes are essential for DNA replication and usually work in pairs to create two identical DNA strands from a single
original DNA molecule.
● DNA polymerase adds nucleotides to the 3'- end of a DNA strand, one nucleotide at a time (Replication Fork).
● Every time a cell divides, DNA polymerases are required to help duplicate the cell's DNA, so that a copy of the original DNA
molecule can be passed to each daughter cell.
● Before replication can take place, an enzyme called helicase unwinds the DNA molecule from its tightly woven form, in the
process breaking the hydrogen bonds between the nucleotide bases. This opens up or "unzips" the double-stranded DNA to
give two single strands of DNA that can be used as templates for replication.
● The lagging strand of the replication fork is transcribed discontinuously, so DNA polymerase is required to form Okazaki
fragments which are joined together by DNA ligase.
● Technology is adapted in the experimental protocol polymerase chain reaction PCR using Taq polymerase.
● Retroviruses e.g. HIV encode an unusual DNA polymerase called reverse transcriptase.

IgM
● is a large polymeric immunoglobulin.
● One of the five major forms of immunoglobulin classes or isotypes – IgM, IgD, IgG, IgE and IgA.
● It’s even better at activating complement than is IgG, and is the first antibody type to appear in the blood after exposure to a
new antigen.
● It is replaced by IgG in a week or two.
● The spleen, where plasmablasts responsible for antibody production reside, is the major site of specific IgM production.
● IgM can bind complement component C1 and activate the classical pathway, leading to opsonization of antigens and cytolysis.
● IgM binds to the polyimmunoglobulin receptor (pIgR) in a process that brings IgM to mucosal surfaces, such as the gut lumen
and into breast milk. This binding depends on J chain.

2015
IFN-g
● Interferon gamma
● The only member of type II interferons
● Critical in innate and adaptive immunity against viral and intracellular bacterial infections
● Promotes cytotoxic cells: NK, Th1 and CTLs
● Predominantly produced by ILCs, NK and NKT cells
● Binds IFNyR to activate JAK-STAT signalling pathways
●
Annexin V
● In flow cytometry, annexin V is commonly used to detect apoptotic cells by its ability to bind to phosphatidylserine, a marker of
apoptosis when it is on the outer leaflet of the plasma membrane.

Respiratory burst
● Respiratory burst (sometimes called oxidative burst) is the rapid release of reactive oxygen species (superoxide radical and
hydrogen peroxide) from different types of cells.
● Neutrophils, monocytes

Histones
● highly alkaline proteins found in eukaryotic cell nuclei that package and order the DNA into structural units called
nucleosomes.
● They are the chief protein components of chromatin, acting as spools around which DNA winds, and playing a role in gene
regulation.
●

AP-1
● Transcription factor
● regulates gene expression in response to a variety of stimuli, including cytokines, growth factors, stress, and bacterial and viral
infections.
● AP-1 controls a number of cellular processes including differentiation, proliferation, and apoptosis.
● Leucine zipper region is responsible for dimerization of the Jun and Fos protein subunits.
● DAG recruits RasGRP which in turn activates Ras which activates MAPK cascade, which activates Fos, a component of AP-1
transcription factor. Activation of MAP kinase Erk allows it to enter the nucleus where it phosphorylates the transcription factor
Elk-1 which then stimulates the transcription of FOS gene. Activated JNK enters the nucleus and phosphorylates c-Jun, which
activates the Jun-Fos dimer for transcription of AP-1.

CTLA-4
● CTLA-4 recruits tyrosine phosphatases that signal through ITIM domains (inhibitory) and inhibit TcR signalling.
● T cells do not secrete IL-2 upon CTLA-4 binding with CD80/86.
● CTLA-4 binds this shared co-receptor with 10-100 times higher affinity than CD28
● This is a regulatory mechanism in place to inhibit autoimmunity.
● Signalling through SHP-2 downregulates the TcR response.
● Ipilimumab

IgM
● is a large polymeric immunoglobulin.
● One of the five major forms of immunoglobulin classes or isotypes – IgM, IgD, IgG, IgE and IgA.
● It’s even better at activating complement than is IgG, and is the first antibody type to appear in the blood after exposure to a
new antigen.
● It is replaced by IgG in a week or two.
● The spleen, where plasmablasts responsible for antibody production reside, is the major site of specific IgM production.
● IgM can bind complement component C1 and activate the classical pathway, leading to opsonization of antigens and cytolysis.
● IgM binds to the polyimmunoglobulin receptor (pIgR) in a process that brings IgM to mucosal surfaces, such as the gut lumen
and into breast milk. This binding depends on J chain.
DAMPs
● Danger Associated Molecular Patterns
● E.g. HMGB1, DNA and RNA, S100 proteins
● Signal through PRRs and initiate a non-infectious inflammatory response.
● Damaged RNAs released from UVB-exposed keratinocytes activate TLR3 on intact keratinocytes. TLR3 activation stimulates
TNF-alpha and IL-6 production, which initiate the cutaneous inflammation associated with sunburn.
● Matzinger to propose the danger hypothesis in 1994, she postulated that the adaptive immune system evolved to respond not
to infection per se but to non-physiological cell death, damage or stress.
● This could explain how adaptive immune responses were stimulated by both infectious and noninfectious agents, such as
tumours and transplanted tissues, all situations in which necrotic cell death would be occurring.
● In the danger model, dying cells were postulated to release endogenous adjuvants that, using similar nomenclature to PAMPs,
have been called damage-associated molecular patterns (DAMPs).
● There are now increasing numbers of examples in which cell death is linked to initiation of an immune response. In
autoimmunity, B-islet cell death induction triggers diabetes. Killing tumour cells via chemotherapy leads to T cell immunity.
Injury to one eye can initiate an autoimmune response that destroys the contralateral eye.
● Adjuvants vaccines: CFA, MPL, MF59

Eosinophils
● Eosinophils are granulocytes that were first described to stain with acid aniline dyes, such as eosin.
● Blood and tissue eosinophilia are hallmark signs of helminth infection, allergy, asthma, eosinophilic gastrointestinal disorders,
and a number of other rare disorders.
● Human eosinophils have a bilobed nucleus with highly condensed chromatin and 2 major types of granules, specific and
primary.
● A major cationic protein in the specific granules are major basic protein (MBP) to activate neutrophils.
● Eosinophils express an array of cell-surface molecules, including immunoglobulin receptors for IgG (FcgRII/CD32) and IgA
(FcaRI/CD89); complement receptors (CR1/CD35, CR3, and CD88); cytokine receptors (IL-3R, IL-5R, and GM-CSF that
promote eosinophil development, as well as receptors for IL-1a, IL-2, IL-4, IFN-a, and TNF-a); chemokines (CCR1 and CCR3);
adhesion molecules (very late antigen 4, a4b7); and TLRs (particularly TLR7/8).
● Eosinophils are activated through receptors to release their cytotoxic granules through piecemeal degranulation.
● Eosinophils develop in the bone marrow and are released into the circulation, most notably after stimulation by IL-5 priming
them to enter the GI or mucosa.
● Eosinophils can express a range of cytokines, their production of cytotoxic granule proteins is thought to be their major effector
function. They secrete IL-4 and IL-13 to activate M1 macrophages and also prime B cells to secrete IgG and recruit Th2 cells.

Therapeutic antibodies
● Bind to block- Ipilimumab
● Bind to kill- Rituximab
● Drug delivery

Type I Interferons
● Type I interferons (IFNs) are a large group of interferon proteins that have diverse effects on innate and adaptive immune cells
during infection with viruses, bacteria, parasites and fungi, directly and/or indirectly through the induction of other mediators.
● Important for host defence against viruses.
 ● The well known mammalian types are IFN-α (alpha), IFN-β (beta).
● Shown to cause immunopathology in some acute viral infections, such as influenza virus infection.
● IFN-β1 is used as a treatment for multiple sclerosis as it reduces the relapse rate. ⅓ patients are unresponsive. It’s
mechanism of action is to inhibit Th1 and Th17 cells, while promoting IL-10 and IL-27 secretion. It’s side effect is flu like
symptoms.

Monoclonal antibodies
● mAb
● are antibodies that are made by identical immune cells that are all clones of a unique parent cell.
● Monoclonal antibodies can have monovalent affinity, in that they bind to the same epitope.
● Discovered by Kohler and Ehrlich
● First monoclonal antibody is Muromonab (anti-CD3) 1986.
● Murine derived mAbs induce HAMA response, leading to the production of chimeric (ending in -ximab) humanised (-zumab)
and fully human antibodies (-umab).
● The steps in monoclonal antibody production include: immunisation of mice to produce antibody and memory B cells, somatic
cell fusion of splenocytes with myeloma cells to form a hybrid, selection and screening for the specific antibodies we want, and
finally subcloning.
● Have many uses including: soluble antigen/cell bound antigen with blocking (e.g. Adalimumab), cell bound inducing depletion
(Rituximab) and as drug delivery carriers (ensuring chemotherapeutic drugs are targeted to cancer cells).

Interleukin 1
● Interleukin 1 (IL-1) is a general name for two distinct proteins, IL-1 alpha and IL-1 beta
● Along with IL-1 receptor antagonist (IL-1ra) and IL-18, these molecules play important roles in the up- and down-regulation of
acute inflammation.
● Although the induction of IL-1 is due to inflammation the effects of IL-1 are not limited to inflammation, it is associated with
bone formation and remodeling, insulin secretion, appetite regulation and neuronal phenotype development.
● Pro IL-1B and IL-18 are cleaved by caspase-1 in the inflammasome.
● Canakinumab a human monoclonal antibody targeted at IL-1B.

Protein Kinase A
● is a family of enzymes whose activity is dependent on cellular levels of cyclic AMP (cAMP).
● also known as cAMP-dependent protein kinase
● PKA is one of the most widely researched protein kinases
● has several functions in the cell, including regulation of glycogen, sugar, and lipid metabolism.
● Extracellular hormones such as glucagon and epinephrine begin an intracellular signalling cascade that triggers protein kinase
A activation by first binding to a G protein–coupled receptor (GPCR) on the target cell.
● The steps in PKA activation are: Cytosolic cAMP increases, Two cAMP molecules bind to each PKA regulatory subunit, The
regulatory subunits move out of the active sites of the catalytic subunits and the R2C2 complex dissociates, The free catalytic
subunits interact with proteins to phosphorylate Ser or Thr residues.
● Protein kinase A acts to phosphorylate many enzymes important in metabolism of hepatocytes.

Complement
● The complement system is a part of the innate immune system that enhances (complements) the ability of antibodies and
phagocytic cells to clear microbes and damaged cells from an organism, promotes inflammation, and attacks the pathogen's
cell membrane.
● Three functions: Phagocytosis by opsonization antigens (C3B), inflammation by attracting macrophages and neutrophils and
membrane attack complex to rupture the cell wall of bacteria.
● Three biochemical pathways activate the complement system: the classical complement pathway, the alternative complement
pathway, and the lectin pathway.
● In the classical pathway, C1 binds with its C1q subunits to Fc fragments (made of CH2 region) of IgG or IgM, which has
formed a complex with antigens.
● In the alternative pathway C3 convertase recruits C5b which recruits and assembles C6, C7, C8 and multiple C9 molecules to
assemble the membrane attack complex. This creates a hole or pore in the membrane that can kill or damage the pathogen or
cell.
● The lectin pathway is homologous to the classical pathway, but with the opsonin, mannose-binding lectin (MBL), and ficolins,
instead of C1q.
● The complement system has the potential to be extremely damaging to host tissues, meaning its activation must be tightly
regulated.

ATP
● Adenosine triphosphate, (ATP), energy-carrying molecule found in the cells of all living things.
● ATP is a nucleotide that consists of three main structures: the nitrogenous base, adenine; the sugar, ribose; and a chain of
three phosphate groups bound to ribose.
● The outer phosphate is removed from ATP to yield energy; when this occurs ATP is converted to adenosine diphosphate
(ADP).
Phosphatidylserine
● is a phospholipid and is a component of the cell membrane.
● It plays a key role in cell cycle signaling, specifically in relationship to apoptosis.
● It consists of two fatty acids attached in ester linkage to the first and second carbon of glycerol and serine attached through a
phosphodiester linkage to the third carbon of the glycerol.
● When the phosphatidylserines flip to the extracellular (outer) surface of the cell, they act as a signal for macrophages to engulf
the cells.
● Phosphatidylserine plays a role in blood coagulation (also known as clotting). When circulating platelets encounter the site of
an injury, collagen and thrombin -mediated activation causes externalization of phosphatidylserine (PS) from the inner
membrane layer, where it serves as a pro-coagulant surface.

TGF-B
● Transforming Growth Factor Beta
● The TGF-β superfamily includes endogenous growth inhibiting proteins.
● An increase in expression of TGF-β often correlates with the malignancy of many cancers and a defect in the cellular growth
inhibition response to TGF-β.
● TGF-β plays a crucial role in the regulation of the cell cycle by blocking progress through G1 phase.
● TGF-β1 plays a role in the induction from CD4+ T cells of both iTregs, which have a regulatory function, and Th17 cells which
secrete pro-inflammatory cytokines.
● TGF-B plays a dual role in anti-tumour immune responses, in early tumour progression it has an anti-tumour tole, but in the
late phases it is pro-tumour.
● TGF-β has been shown to function as a chemoattractant as well as an upregulator of inflammatory response for monocytes.
● TGF-B is secreted by tolerogenic DCs and Foxp3+ Tregs
● Higher concentrations of TGF-β are found in the blood and cerebrospinal fluid of patients with Alzheimer's disease as
compared to control subjects, suggesting a possible role in the neurodegenerative cascade leading to Alzheimer's disease
symptoms and pathology.

Caspases
● Cysteine proteases guided by aspartate residues
● Key to apoptosis and production of IL-1B and IL-18 in the inflammasome
● Form a large family
● Inflammation caspases: 1,4,5,11
● Apoptosis initiators: 2,8,9,10 and executioners: 3,6,7
● Pro-domain and a small subunit linked to a large subunit

Perforin
● Perforin is a pore forming cytolytic protein found in the granules of cytotoxic T lymphocytes (CTLs) and NK cells.
● Upon degranulation, perforin binds to the target cell's plasma membrane, and oligomerises in a Ca2+ dependent manner to
form pores on the target cell.
● The pore formed allows for the passive diffusion of a family of pro-apoptotic proteases, known as the granzymes, into the
target cell.
● Perforin has structural and functional similarities to complement component 9 (C9).

Reverse vaccinology
● Vaccinomics is a new branch of bioinformatics that deals with designing a candidate vaccine against a pathogen that can be
used for production of the vaccine in less time as that of conventional vaccinology.
● Reverse vaccinology is a part of vaccinomics which starts with the genome of pathogen and is used for the predicting the
epitope.
● Epitope prediction is the heart of reverse vaccinology.
● Reverse vaccinology was used for designing vaccines against some diseases eg. Malaria, Anthrax, Endocarditis, Meningitidis
etc.

Th17
● 95% of T cells = ab T cells
● Production of Il-1b, Il-6, TGF-B, IL-23 promotes the differentiation of Th17 cells vis STAT3 - RORt activation (Dectin 1 binding
= example of such activation of DCs.
● IL-1, IL-1R, NLRP3, caspase 1 are all reguired for the induction of Th17 (KO mice display less)
● Th17 cells produe IL-17A,F, IL-21, IL-22, GM-CSF.
● IL-10 production has been observed, perhaps as control mechanism
● IL-17 activates endothelial and epithelial cells to produce chemokines and pro inflammatory mediatiors that promote the
recruitment of neutrophils to site of insult. (IL-6, IL-8 induction etc)
● IL-22 stimulates production of AMPs by epithelial cells, and increases proliferation rate, crucial for barrier integrity.
● The expression of pIgR by IECs is IL-17 dependant, therefore lack of IL-17 in gut leads to accumulation of IgA in lamina
propria, diminished S-IgA in lumen.
● Th17 therefore from these mechanisms are crucial for mucosal immunity.
 ● Segmented Filamentous bacteria (SFBs) shown to promote Th17 immunity, protective in S.aureus mediated pneumonia.
(Possibly NLR signalling at play)
● Extracellular bacteria and fungi immunity, especially Candida Albicans.
● Role in pertussis also, Th17 KO - increase CFUs (Synergistic clearance with Th1)
● Alum can induce Th17 ( probably due to localised cell damage, subsequent Il-1B etc)
● However prominent mediators of autoimmunity
● Th17 can be used to transfer autoimmunity
● IL-17 KO attenuates EAE, RA, Colitis
● Secukinumab = Anti-IL-17A --- Psoriasis excellent profile ( appears to not compromise Th17 immunity also)

Immunological Synapse
● is the interface between an antigen-presenting cell or target cell and a lymphocyte such as an effector T cell or Natural Killer
cell.
● The initial interaction occurs with adhesion molecules such as ICAM:LFA-1, allowing T cells to bind the target cell and extend
it’s pseudopodia to scan the surface of the cell for MHC:peptide complex.
● The immunological synapse formation begins when the T-cell receptor (TCR) binds to the peptide:MHC complex on the
antigen-presenting cell, activating downstream signalling pathways through TcR ITAM domain.
● Actin polymerization and reorganisation promotes clustering of receptors to amplify the response.

2010
Receptor Mediated Endocytosis
● Also called clathrin-mediated endocytosis, is a process by which cells absorb metabolites, hormones, other proteins – and in
some cases viruses – by the inward budding of plasma membrane vesicles containing proteins with receptor sites specific to
the molecules being absorbed.
● Clathrin-mediated endocytosis of many receptor types begins with ligand binding its receptor. The ligand and receptor
(sometimes bound to an adaptor protein) then diffuse through the plasma membrane until captured by a preformed or forming
clathrin-coated pit.
● A mature pit will pinch off from the plasma membrane forming a clathrin-coated vesicle that then uncoats and typically fuses to
an early endosome. Once fused the endocytosed cargo (receptor and/or ligand) can then be sorted to lysosomal, recycling, or
other trafficking pathways.
● It is widely used for the specific uptake of certain substances required by the cell (examples include LDL via the LDL receptor
or iron via transferrin).

Aspirin
● Also known as acetylsalicylic acid (ASA), is a medication used to treat pain, fever, or inflammation.
● First synthesised by BAYER.
● Aspirin is a nonsteroidal anti-inflammatory drug (NSAID) and works similar to other NSAIDs but also suppresses the normal
functioning of platelets.
● Cox inhibitor (acetylates key amino acid in active site) leading to decreased prostanglandins production, application to chronic
diseases was affected due to effects on CoX1 (good enzyme, stomach etc). Saw risk of cardiac dysfuction
● Aspirin is one of the most widely used medications globally, with an estimated 40,000 tonnes (44,000 tons) (50 to 120 billion
pills) consumed each year.
● Aspirin is used in the treatment of a number of conditions, including fever, pain, rheumatic fever, and inflammatory diseases,
such as rheumatoid arthritis.
● Aspirin is thought to reduce the overall risk of both getting cancer and dying from cancer. This effect is particularly beneficial
for colorectal cancer (CRC) but must be taken for at least 10–20 years to see this benefit.
● 2nd leading cause of death from cancer is increased clotting - aspirin application
● Role in prevention/ inhibiting inflammaging - contributing to decreased risk of some diseases.
● Baby aspirin dementia ( decreased accumulation of plaques)
● Celebrex and Vioxx = Cox 2 specific drugs

T-Bet
● T-box transcription factor
● an essential regulator of effector differentiation and function in multiple different immune lineages, including CD4, CD8, NKT,
ILC (including NK) and B cells
 ● Inflammatory cytokines induce T-bet expression in a graded manner, which in turn regulates distinct differentiation outcomes.
For example, T-bet can act as a fulcrum between Th1 and Tfh cell differentiation, pathogenic and non-pathogenic Th17 cells,
and CD8 effector and memory T cells
● During infection, T-bet induces CXCR3 expression on multiple different lymphocytes. This promotes an effective immune
response by allowing the colocalization of multiple cell types within the appropriate tissues for targeted effector function.
● T-bet and Eomes coregulate many genes, but have opposing functions in memory development.

anti-TNFa (asked as just TNF-a before)

● Tumour Necrosis Factor
● TNF is involved in autoimmune and immune-mediated disorders such as rheumatoid arthritis, ankylosing spondylitis,
inflammatory bowel disease, psoriasis, hidradenitis suppurativa and refractory asthma, so TNF inhibitors may be used in their
treatment.
● The important side effects of TNF inhibitors include lymphomas, infections (especially reactivation of latent tuberculosis),
congestive heart failure, demyelinating disease, a lupus-like syndrome, induction of auto-antibodies, injection site reactions,
and systemic side effects.

Polysaccharide vaccines
● PS vaccines, such as those developed against Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae
type b, and Salmonella typhi, prevent infection by inducing an immune response against specific capsular polysaccharides.
● Many bacteria are surrounded by a polysaccharide capsule, which both provides the antigens against which antibodies can
act, the molecules that largely determine how virulent and pathogenic the organism and protects the main bacterial cell from
the body's defense systems.
● It is relatively easy to create polysaccharide vaccines against the molecules in the capsule; but these molecules are often
small, and not very immunogenic. As a consequence they: tend not to be effective in infants and young children (under 18-24
months), induce only short-term immunity - there is a slow immune response, with antibody levels rising slowly, with no
immune memory.
● The development of glycoconjugate vaccines overcame many limitations associated with PS vaccines by eliciting a
quantitatively and qualitatively different immune response.

Peyer’s patch
● Small masses of lymphatic tissue
● They are an important part of gut associated lymphoid tissue usually found in humans in the lowest portion of the small
intestine, mainly in the distal jejunum and the ileum, but also could be detected in the duodenum.
● Also known as aggregated lymphoid nodules.
● Special epithelial cells known as microfold cells line the side of the Peyer’s patch facing the intestinal lumen, while the outer
side contains many lymphoid cells and lymphatic vessels.
● Antigens from microbes in the gut are absorbed via endocytosis by microfold cells lining the surface of each Peyer’s patch.
These antigens are passed on to the lymphoid tissue, where they are absorbed by macrophages and presented to T
lymphocytes and B lymphocytes.
● About 100 are found in humans.

IgE
● Immunoglobulin E are an antibody isotype synthesized by plasma cells.
● Shortest half life of all antibodies and lowest serum concentration
● is designed to attach to mast cells with high affinity via FCeRI in tissues, and basophils with high affinity i circulation. These
cells are said to be sensitiseD upon IgE binding. IgE binding also upregulates further FceRI expression. Upon cognate antigen
exposure (Challenge) there is aggregation of these receptors and cell activation via ITAMs - Syk, it will cause the mast cells
and basophils to degranulate releasing powerful mediators such as prostaglandins, leukotrienes, and cytokines, and release
its granules which contain powerful mediators of inflammation like histamine.
● ILC2 cells provide source of IL-13 which leads to recruitment of basophils. These basophils produce IL-4 and IL-13 which
promote IgE CSR. Th2 cells amplify IgE CSR later on. CD40L essential too.
● Hyper IgE syndrome - defect in CD40/CD40L
● IgE can upregulate the expression of both types of Fcε receptors: low (FceRII (DCs, macrophages) or high affinity (FceRI).
High affinity receptor is also found on basophils and dendritic cells.
● IgE has a role in Type I Hypersensitivity: (initiation and propagation of response. mast cell mediators produce the symptoms of
allergy, which range from hay fever and hives to asthma and anaphylactic shock, depending on the site of antigen entry and
dose. Increased permeability can lead to edema.
● Upon binding of IgE, basophils release Type 2 cytokines: IL-4, IL-13.
● Involved in various allergic diseases, such as allergic asthma, most types of sinusitis, allergic rhinitis, food allergies, and
specific types of chronic urticaria and atopic dermatitis.
● 50% of asthma in adults is allergic asthma - see increased mucus production, bronchoconstriction etc
● The real role of IgE is in resistance to parasites, such as worms. Hygiene Hypothesis.
● Anti-IgE antibody as asthma therapy - omalzimab - shows promise

Leucine Zipper
 periodic repetition of a leucine residue at every seventh position (heptad repeat) and forms an α‐helical conformation
 facilitates dimerisation and in some cases higher oligomerisation of proteins by forming a parallel helix–helix association
stabilised by formation of an interhelical hydrophobic core involving leucine side chain
 The ‘basic region ZIP’ (bZIP motif) is a fusion between a segment that is rich in basic residues facilitating DNA binding and
ZIP, forming a class of eukaryotic transcription factors.
 The DNA binding specificity of bZIP is determined by the basic region that directly binds a class of palindromic DNA
sequences.
 ZIP‐related sequence motifs containing an incomplete heptad repeat are frequently found in a variety of proteins and catalyse
dimerisation/oligomerisation by forming both parallel and antiparallel coiled coils.